Microparticles and nanoparticles made up of hydrophobized polysaccharides and an alpha-cyclodextrine

ABSTRACT

Microparticles and nanoparticles of hydrophobized polysaccharide and an alpha-cyclodextrin, obtained by self-association in an aqueous medium, the hydrophobized polysaccharide being obtained by grafting of alkyl chains derived from fatty acids, by an acylation reaction. These microparticles and nanoparticles constitute systems used for encapsulating substances of interest, in particular in the pharmaceutical field, and vectorization thereof for therapeutic purposes.

The invention relates to microparticles and nanoparticles consisting ofhydrophobized polysaccharides and an alpha-cyclodextrin.

The invention also relates to the use thereof as an encapsulationsystem.

At present, particles capable of containing and of vectorizing orencapsulating an active ingredient are the subject of active research.These particles must be capable of trapping a substance of interest, ofconveying it in the body to the target cell or tissue, and thenreleasing it without any change in its structure. It is important thatthese particles are not toxic. However, there is a considerable drawbackassociated with their preparation. In fact, the latter often requires,in one step at least, the use of an organic solvent and/or the use ofsurfactants that are not biocompatible, sometimes under conditions ofvery high acidity. Removal of the molecules of solvent and/or ofsurfactant is often lengthy and incomplete, and adds a cost to theproduction of the particle. The toxic residual traces may moreovercontribute to degradation of the active substances immobilized in theparticles. This is why it is sought to produce these particlespreferably in an aqueous medium, starting from polymers that arebiocompatible and biodegradable.

The polysaccharides form a very useful class of polymers in the field ofencapsulation. These polysaccharides include chitosan, chitin,hyaluronic acid and the glycosaminoglycans (GAGs).

Chitosan is a linear heteropolymer of N-acetyl-D-glucosamine andD-glucosamine joined together at β (1-4) according to the formula:

in which

-   -   m represents the number of D-glucosamine units,    -   n represents the number of N-acetyl-D-glucosamine units,        provided that the percentage of m relative to the total number        of units is greater than 50%. It has the advantage of being        biocompatible and mucoadhesive. Chitosan nanoparticles have been        used as adjuvants for vaccination by the mucosal route in        animals (Annales de Medecine Vétérinaire, 2003, 147, 343-350).        An adjuvant is a substance which, when it is administered at the        same time as an antigen, increases the immune response to this        antigen. The advantage of vaccination by the mucosal route is        the introduction of an immune response at the point of entry of        microbes. Vaccines administered alone by the mucosal route have        a low bioavailability. They must be co-administered with        substances that promote their penetration or with adjuvants.

Chitin is a linear heteropolymer of N-acetyl-D-glucosamine andD-glucosamine joined together at β (1-4) according to the formula:

in which

-   -   m represents the number of D-glucosamine units,    -   n represents the number of N-acetyl-D-glucosamine units,        provided that the percentage of m relative to the total number        of units is below 50%. Chitin is a powerful hydrating agent, an        effective scavenger of heavy metals, which are responsible for a        great many contact allergies. It is used in the treatment of        burns as it has wound-healing properties. Chitin is also used        for filtering wastewater: it forms ionizable chains, allowing        fixation of organic elements. It is also used in the food        industry (manufacture of juice), as a processing aid.

One of the first steps in the colonization of biotic surfaces bymicrobes involves their interaction with receptors located at cellularlevel such as the glycosaminoglycans (GAGs), in particular heparin,heparan sulphate, hyaluronic acid, dermatan sulphate, keratan sulphateand chondroitin sulphate. Numerous viruses, bacteria, fungi andparasites are capable of binding to the GAGs present on the surface ofthe cells, thus facilitating their initial attachment and then entryinto the cell followed by infection. Data from the literature indicatethat the inhibitory effect of heparin, heparan sulphate, dermatansulphate and chondroitin sulphate has been demonstrated on the humanherpes simplex virus (HSV) (Journal of Bacteriology, 1964, 87(5),1060-1066, Virology. 1995, 208(2), 531-539, Journal of Virology. 1989,63(1), 52-58), whereas the binding of particles imitating the humanpapillomavirus (HPV) to cells was inhibited by heparin (The Journal ofBiological Chemistry. 1999, 274(9), 5810-5822). Moreover, heparansulphate has been found to play an important role in the prevention ofinfections by HPV-16 (Journal of Virology. 2009, 83(5), 2067-2074).

Heparin coating of abiotic surfaces has enabled bacterial adhesion to bereduced by 90% (Journal of Urology. 1987, 138, 423-426). GAGs and thesulphated polysaccharides have shown antibacterial activity against:Staphylococcus aureus, Gardnerella vaginalis, Mycobacteriumtuberculosis, Listeria monocytogenes, Neisseria gonorrhoeae,Helicobacter pylori, Yersinia enterocolitica, Mycoplasma pneumoniae,Streptococcus mutans, Chlamydia trachomatis, Staphylococcus aureus,Escherichia coli, Pseudomonas aeruginosa (US 2005/0203055).

Heparin coating of abiotic surfaces has made it possible to reduce theadhesion of Candida albicans by approximately 50% (Infection andImmunity. 1993, 61(11), 4560-4568).

US patent 2005/0203055 has shown that cellulose sulphate has:

-   -   antiviral properties against human immunodeficiency virus (HIV),        HSV, HPV, bovine papillomavirus (BPV)    -   antiparasitic properties against Trichomonas vaginalis,    -   antifungal properties against Aspergillus niger and Candida        albicans.

Other sulphated polysaccharides such as cellulose sulphate, carrageenan,dextran sulphate, and dextrin sulphate have shown antimicrobialactivity.

Hyaluronic acid helps to protect the joints by increasing the viscosityof synovial fluid and by making cartilage more elastic. Hyaluronic acidis the only non-sulphated GAG. Hyaluronic acid is a natural constituentof the dermis and plays an important role in hydration, tonicity andelasticity of the skin.

Polysaccharides grafted with hydrophobic chains are amphiphilic systemscapable of spontaneous self-association in an aqueous medium in the formof micelles of the core-corona type that can accommodate an activeingredient (Drug Discovery Today, 2012, 17, 623-629). However, theirsolubility in aqueous media decreases owing to the presence of thehydrophobic grafted chains.

The use of cyclic polysaccharides, such as cyclodextrins, leads to theformation of particles constituting inclusion systems that are solublein aqueous media. These systems, of variable size and structure, rangingfrom nanoparticle to hydrogel, are capable of vectorization a substanceof interest.

Gref et al. (US2005/004348A1) describe particles obtained by graftingone or more (for example two) cyclic polysaccharides, such asbeta-cyclodextrin, onto a biodegradable polymer such aspoly(ε-caprolactone) and use thereof as vectors of active substances. Inthis case, the cyclic polysaccharide is bound covalently to thebiodegradable polymer. This same patent describes the formation ofnanoparticles by mixing a dextran bearing lauryl chains and abeta-cyclodextrin polymer.

Bochot et al. (US2006/0188464A1; EP1590077B1; International Journal ofPharmaceutics, 2007, 339, 121) describe, in their publication and theirapplications, beads varying in size from 1 to 3 mm, obtained by mixingan oil and a solution of alpha-cyclodextrin. They do not use linearpolysaccharides described above and, moreover, the particle size formedis in the millimetre range.

One of the aims of the invention is to supply inclusion complexesbetween a polysaccharide and a cyclodextrin.

Another aim of the invention is to propose microparticles ornanoparticles formed from the aforesaid inclusion complexes.

Another aim is to supply a simple method for the preparation of saidparticles without necessarily using surfactants or organic solvents.

In addition, the invention relates to the use of said particles as such,without necessarily adding substance(s) of interest.

The invention relates to the use of said particles for encapsulating oneor more substance(s) of interest.

The invention relates to an inclusion complex formed between:

-   -   a polysaccharide comprising hydrophobic groups bound covalently        to said polysaccharide,    -   a cyclodextrin (CD) in the form of monomer, the polysaccharide        and the cyclodextrin being bound non-covalently.

According to another advantageous aspect, the invention relates to aninclusion complex formed by the interaction between:

-   -   a polysaccharide comprising hydrophobic groups bound covalently        to said polysaccharide,    -   and a cyclodextrin (CD) in the form of monomer, the        polysaccharide and the cyclodextrin being bound non-covalently.

The term “inclusion complex” denotes a system that results from theinteraction of a “host” molecule that allows within its cavity one ormore other “guest” molecules without any covalent bond beingestablished.

The term “polysaccharide” denotes a carbohydrate macromolecule formed bythe chain formation of a large number of elementary sugars. The majorityof the polysaccharides are hydrophilic, they carry —OH and/or —NH₂,and/or —COOH, and/or —CH₂—OH, and/or —SO₃ ⁻ groups, or groups derivedfrom the latter. The nature of these groups allows them to bedifferentiated from a structural standpoint and from the standpoint ofthe physicochemical and biological properties. In this case the term“polysaccharide” denotes a linear or branched polysaccharide.

The expression “polysaccharide comprising hydrophobic groups” means thatthe polysaccharide has been “hydrophobized” by grafting, on the —OHand/or —NH₂, and/or —COOH, and/or —CH₂—OH, and/or —SO₃ ⁻ groups, ofalkyl chains that are naturally hydrophobic owing to their non-polarcharacter. The “polysaccharide comprising hydrophobic groups” istherefore an amphiphilic polysaccharide. Under certain conditions, thesepolysaccharides are capable of self-association, forming micelles of thecore-corona type in aqueous media (Drug Discovery Today, 2012, 623-629).

A “cyclodextrin” (or cycloamylose) is a cyclic oligosaccharide ofβ-D-glucopyranose joined together by α(1-4) bonds. It is a cage moleculeof natural origin that makes it possible to encapsulate variousmolecules, in particular molecules of therapeutic interest. There arevarious sizes, each having the shape of a “lampshade”. It bearshydrophilic groups (—OH) located on the outside, the assembly delimitinga relatively hydrophobic cavity. This amphiphilic character allowscyclodextrin to enclose hydrophobic molecules within its cavity, to formwater-soluble inclusion complexes. Its biodegradable nature predisposesit to important applications in the areas of agri-food, cosmetics andpharmaceuticals. Encapsulation in cyclodextrins in fact makes itpossible to protect fragile molecules or ensure slow and controlledrelease thereof.

Using alpha-cyclodextrin instead of a cyclodextrin polymer represents anobvious advantage from the economic and regulatory standpoint as thiscyclodextrin is commercially available and is a recognizedpharmaceutical excipient, accepted by the majority of pharmacopoeias.The expression “the polysaccharide and the cyclodextrin being boundnon-covalently” means that the interactions between these two moleculesare van der Waals bonds and/or hydrogen bonds, and/or electrostaticbonds, and/or hydrophobic bonds, and not covalent bonds.

These inclusion complexes are thus formed exclusively by non-covalentbonds by simple mixing of alpha-cyclodextrin and polysaccharide graftedwith hydrophobic groups. Thus, by using the same procedure and byvarying the type of these non-covalent interactions, particles of variedsize and structure may be formed.

The invention thus relates to an inclusion complex formed by theinteraction between at least:

-   -   a polysaccharide selected from chitosan, dextran, hyaluronic        acid, amylose, amylopectin, pullulan, heparin, chitin, heparan        sulphate, dermatan sulphate, keratan sulphate, chondroitin        sulphate, cellulose sulphate, dextran sulphate, dextrin        sulphate, starch, pectin, the alginates, the carrageenans,        fucan, curdlan, xylan, polyguluronic acid, xanthan, arabinan,        polymannuronic acid, and derivatives thereof, said        polysaccharide comprising hydrophobic groups selected from        linear or branched alkyl groups containing from 2 to 1000 carbon        atoms, or linear or branched, in particular linear, alkenyl        groups, which may contain at least one C═C double bond, said        hydrophobic groups being bound covalently to said        polysaccharide,    -   and an α-cyclodextrin (CD) in the form of monomer, the        polysaccharide and the cyclodextrin being bound non-covalently.

Advantageously, in the inclusion complex according to the invention, thepolysaccharide is made up of at least 3 saccharide units, its molecularweight being in particular in the range from 100 Da to 1,000,000 kDa,and in particular is equal to 20 kDa, 145 kDa or 250 kDa.

According to another aspect, in the inclusion complex according to theinvention, the polysaccharide is made up of at least 3 saccharide units,its molecular weight being in particular in the range from 5 kDa to100,000 kDa, and in particular is equal to 20 kDa, 145 kDa or 250 kDa.

Advantageously, the ratio of the concentration of the cyclodextrin tothe concentration of the polysaccharide is in the range from 10⁻⁶ to900,000, in particular in the range from 4 to 15, and in particular isequal to 10.

According to another aspect, the ratio of the concentration of thecyclodextrin to the concentration of the polysaccharide is in the rangefrom 0.01 to 1500, in particular in the range from 4 to 15, and inparticular is equal to 10.

This parameter is very important as it makes it possible to modulate theparticle size obtained from the aforesaid inclusion complex by alteringthe ratio of the concentration of the cyclodextrin to that of thepolysaccharide.

The average particle size might increase when the ratio of theconcentration of the cyclodextrin to the concentration of thepolysaccharide decreases.

If the ratio is less than 0.01, no particles are formed. If the ratio isgreater than 20, particles are formed. Above a concentration ratio of1500, and in particular for a concentration above 50 g/L,alpha-cyclodextrin is no longer water-soluble.

The solubility of the polysaccharide is not a limiting factor information of the particles, as the latter form even if thepolysaccharide is used in the form of a suspension in water. Being ableto use a polysaccharide in the form of suspension for formulatingnanoparticles and microparticles has the advantage thatmore-concentrated particles are obtained. With this new technology,polysaccharides that were difficult to formulate as nanoparticles ormicroparticles on account of their problems of solubility in water (inparticular chitin) thus find new applications.

Advantageously, this method is simple, and it reduces pollution of theenvironment, because it is not essential to use solvents, surfactants orreagents. It also lowers energy consumption. It does not necessarily usea heating step if a purification step is carried out after theirpreparation.

Advantageously, the polysaccharides retain their properties, inparticular antimicrobial, even after they are formulated asnanoparticles and microparticles.

The invention relates in particular to an inclusion complex in which thepolysaccharide is selected from chitosan, dextran, hyaluronic acid,amylose, amylopectin, pullulan, heparin, chitin, cellulose derivatives,heparan sulphate, dermatan sulphate, keratan sulphate, chondroitinsulphate, cellulose sulphate, dextran sulphate, dextrin sulphate,starch, pectin, the alginates, the carrageenans, fucan, curdlan, xylan,polyguluronic acid, xanthan, arabinan, polymannuronic acid, andderivatives thereof, and is in particular chitosan.

They may be neutral (dextran for example), or may have an overallpositive charge (chitosan for example) or negative charge (heparin,hyaluronic acid, pectin).

In a particular aspect of the invention, the polysaccharides haveintrinsic properties even without adding active molecules. Chitosan hasthe advantage of being biocompatible and mucoadhesive, and may be usedas adjuvant for vaccination by the mucosal route. Chitin has hydratingand wound-healing properties and is able to capture heavy metals andpurify wastewater. The GAGs and the sulphated polysaccharides haveactivity for preventing, inhibiting and/or treating fungal, bacterial,viral and/or parasitic infections on biotic or abiotic surfaces.Hyaluronic acid is known to promote hydration, tonicity and elasticityof the skin and to protect the joints by increasing the viscosity of thesynovial fluid and by making the cartilage more elastic.

These polysaccharides are able to prevent and inhibit the formation ofbiofilms. A biofilm is defined as an organized community of cells fixedto a biotic or abiotic surface, incorporated in extracellular material.The biofilms are the predominant form of life of microorganisms andconstitute a means by which the latter display resistance toantimicrobial agents. They can colonize biotic surfaces such as thesurface of cells, tissues or organs (for example the skin and thebuccal, nasal, ocular, aural, vaginal, and rectal mucosae and/or thedigestive system) or abiotic surfaces such as plastic, glass, metal orany other material on which microorganisms can develop.

According to another particular embodiment, in the inclusion complex ofthe invention, the degree of substitution of the polysaccharide withhydrophobic groups is from 0.001 to 100%, in particular from 0.05 to50%.

According to another particular embodiment, in the inclusion complex ofthe invention, the degree of substitution of the polysaccharide withhydrophobic groups is from 0.1 to 70%, in particular equal to 2%, 13% or17%.

The degree of substitution reflects the number of hydrophobic groupsbound to 100 saccharide units of the polysaccharide chain. It isdetermined by the experimental conditions of grafting and can bemeasured by nuclear magnetic resonance (NMR) or by elemental analysisfor example.

It is also a parameter allowing the average particle size formed fromthe inclusion complexes to be modulated. When the degree of substitutionincreases, their average size may decrease or increase, depending on thepolysaccharide used.

According to a particular embodiment, in the inclusion complex of theinvention, the hydrophobic groups are linear or branched, in particularlinear, alkyl groups containing from 2 to 1000 carbon atoms, or linearor branched, in particular linear, alkenyl groups, which may contain atleast one C═C double bond.

According to another particular embodiment, in the inclusion complex ofthe invention, the hydrophobic groups are linear or branched, inparticular linear, alkyl groups containing from 2 to 20 carbon atoms, orlinear or branched, in particular linear, alkenyl groups containing 2 to20 carbon atoms and bearing from 1 to 4 C═C double bonds, conjugated ornot.

According to a particular embodiment, the hydrophobic groups are notaromatic groups.

The fatty acids used for grafting the hydrophobic chains on thepolysaccharide are in particular lauric acid, palmitic acid, oleic acid,stearic acid, linoleic acid, this list in no case being exhaustive andlimiting.

Fatty acid Formula Lauric acid (12:0)

Palmitic acid (16:0)

Stearic acid (18:0)

Linoleic acid (18:2 Δ9, 12)

Linolenic acid (18:3 Δ9, 12, 15)

Oleic acid (18:1 Δ9)

According to a particular embodiment, in the inclusion complex of theinvention, the hydrophobic groups are fixed covalently to thepolysaccharide by a nitrogen atom of said polysaccharide.

This applies to polysaccharides comprising amino groups, in particularchitosan and heparin. These amino groups may undergo a reaction ofN-acylation by reaction with a fatty acid or a fatty acid derivativesuch as a fatty acid chloride or a fatty acid anhydride.

According to another particular embodiment, in the inclusion complex ofthe invention, the hydrophobic groups are fixed covalently to thepolysaccharide by one or more oxygen atoms of said polysaccharide.

According to another particular embodiment, in the inclusion complex ofthe invention, the hydrophobic groups are fixed covalently to thepolysaccharide by one or more oxygen atoms of said polysaccharide, andin particular at the level of the carboxyl function COOH, and/or of agroup CH₂—OH and/or —SO₃ ⁻ of said polysaccharide.

There are many —OH groups on the polysaccharides. They react with thefatty acids or fatty acid derivatives such as the acid chlorides and theacid anhydrides, to give esters. The hydrophobic group of the fatty acidor fatty acid derivative is therefore grafted to the polysaccharide byone of its oxygen atoms, in the form of an acyl group: this is an“O-acylation” reaction.

The O-acylation reaction leads to the formation of ester bonds that areeasily degraded by esterases after administration in vivo.

According to another embodiment of the invention, in the inclusioncomplex of the invention, the hydrophobic groups are fixed covalently tothe polysaccharide by a nitrogen, phosphorus or sulphur atom and byoxygen atoms of said polysaccharide in the proportions from 0.001 to100%.

According to another embodiment of the invention, in the inclusioncomplex of the invention, the hydrophobic groups are fixed covalently tothe polysaccharide by a nitrogen, phosphorus or sulphur atom and byoxygen atoms of said polysaccharide in the proportions from 0.5 to 20%.

When the polysaccharide contains amino groups and hydroxyl groups, byreaction with fatty acids or fatty acid derivatives such as acidchlorides and acid anhydrides, it may undergo both N-acylation andO-acylation. However, the amino groups are more reactive than thehydroxyl groups.

When methanesulphonate is used, O-acylation of chitosan is predominant.

According to another embodiment, in the inclusion complex of theinvention, the cyclodextrin CD has the formula:

in which

-   -   p is an integer equal to 6,    -   —R′, R² and R³, which may be identical or different, in        particular identical, are hydrogen atoms, alkyl groups        comprising 1 to 3 carbon atoms, selected from methyls, ethyls,        propyls, isopropyls, —NH₂ amino groups, —NH₃ ⁺ ammonium groups,        or —SO₄ ²⁻ sulphate groups, and are in particular hydrogen atoms        or methyl groups, said CD being alpha-cyclodextrin (α-CD) in the        form of monomer.

The “cyclodextrins” (CD) are cyclic oligomers of β-D-glucopyranosesjoined together by α(1-4) bonds. Three families are mainly used. Theyare the α—, β- and γ-cyclodextrins formed respectively from 6, 7 or 8glucopyranose subunits.

p=6 corresponds to α-cyclodextrin; p=7 corresponds to β-cyclodextrin andp=8 corresponds to γ-cyclodextrin. They are shown diagrammaticallybelow:

Their geometry is comparable to a truncated cone delimiting a cavity atits centre. It is described in FIG. 15.

They are therefore cage molecules, capable of receiving molecules byinclusion, species in particular of a hydrophobic nature. The size ofthe cavity depends on the nature of the cyclodextrin. The internalportion of the cavity is hydrophobic, and the external portion ishydrophilic.

The —OH groups may be substituted, in particular with methyl,hydroxypropyl or sulphobutyl groups. The substitutions may increase thesolubility of the cyclodextrin. The only cyclodextrin used in theinvention is α-cyclodextrin. The advantage of this cyclodextrin isconnected with its small size, which allows it to interact with thehydrophobic chains bound to the polysaccharide.

According to a particular embodiment, in the inclusion complex of theinvention, the cyclodextrin is functionalized with a ligand selectedfrom antibodies, antibody fragments, receptors, lectins, biotin orderivatives thereof.

The term “ligand” denotes a molecule capable of binding covalently toCD.

The ligand is selected from proteinaceous compounds in particularinvolved in the recognition and/or neutralization of pathogenic agents(antibodies or antibody fragments, receptors, lectins), involved in themetabolism of fatty acids (biotin and derivatives).

According to another particular aspect, in the inclusion complex of theinvention, the cyclodextrin is charged or uncharged.

According to yet another aspect, in the inclusion complex of theinvention, the cyclodextrin is substituted or unsubstituted.

By “substituted cyclodextrin” is meant for example a cyclodextrinsubstituted with an alkyl group, for example a methylated cyclodextrin,with a hydroxyalkyl group, with a maltosyl group, with a galactosylgroup, or with any other molecules.

According to another particular embodiment of the invention, in theinclusion complex:

-   -   the polysaccharide is a chitosan bearing hydrophobic groups,    -   the cyclodextrin is α-CD.

Chitosan, a linear polysaccharide of natural origin, composed randomlyof D-glucosamine deacetylated units bound at β-(1-4) toN-acetyl-D-glucosamine acetylated units, is produced by chemical orenzymatic deacetylation of chitin. Chitosan is biocompatible andbiodegradable. It is non-toxic, and bio-adhesive owing to interactionbetween the positive charges in acid medium (pH<6.0-6.5) carried by theamine functions and the negative charges carried by the biologicalmembranes. It also displays antibacterial and antiviral activity.

Chitosan is soluble in an aqueous solution of acetic acid at lowconcentration by protonation of the amino groups present. The glucosidechain of chitosan is essentially hydrophilic. However, it is possible tograft groups, in particular hydrophobic groups, on the amino andhydroxyl groups. The polysaccharide then becomes amphiphilic and canform micelles, nanoparticles but also hydrogels at higher concentrationby self-association in aqueous media. It is therefore possible toencapsulate active ingredients in the objects that it forms. It may beused for encapsulating active ingredients (paclitaxel, ibuprofen, etc.).

According to another embodiment, in the inclusion complex of theinvention, chitosan bears hydrophobic groups grafted at the level ofcertain nitrogen atoms and has the formula:

in which

-   -   m represents the number of deacetylated units,    -   n represents the number of acetylated units, provided that the        degree of deacetylation (DDA) representing the percentage of m        relative to the total number of units is greater than 50%,    -   R⁴ represents the hydrophobic group and is selected from:        -   a linear or branched alkyl group containing from 1 to 20            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 20 carbon            atoms and bearing from 1 to 4 C═C double bonds, conjugated            or not, in particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups.

According to another embodiment, in the inclusion complex of theinvention, chitosan bears hydrophobic groups grafted at the level ofcertain nitrogen atoms and has the formula

in which

-   -   m represents the number of D-glucosamine units,    -   n represents the number of N-acetyl-D-glucosamine units,        provided that the degree of deacetylation (DDA) representing the        percentage of m relative to the total number of units is greater        than 50%,    -   R⁴ represents the hydrophobic group and is selected from:        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and containing at least one C═C double bond, in            particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups.

DDA of 50% denotes the boundary between chitin and chitosan: when theDDA is below 50%, it is called chitin, otherwise it is called chitosan.

The —(CH₂)₁₄—CH₃ group is derived from palmitic acid, —(CH₂)₁₆—CH₃ isderived from stearic acid, —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ is derived fromlinoleic acid, —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ is derived from oleic acid.

The N-acylated chitosan is obtained from a reaction of the N-acylationtype in the presence of a coupling agent, EDCI(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), which reacts with thecarboxyl groups of the fatty acid (oleic or palmitic for example) toform an intermediate ester capable of binding to the free aminofunctions of the chitosan (Lee, K. Y., et al., Structural Determinationand Interior Polarity of Self-Aggregates Prepared from DeoxycholicAcid-Modified Chitosan in Water, Macromolecules, 1998, 31, 2, 378-383).The following diagram shows the succession of reactions carried out inorder to obtain an N-acylated chitosan, grafted with chains derived fromoleic acid. The same procedure is followed with the other fatty acidssuch as palmitic acid for example.

The IR and NMR spectra of the grafted chitosans reveal the secondaryamine function obtained after N-acylation and the presence of thegrafted alkyl chains.

According to another embodiment of the invention, in the inclusioncomplex, chitosan bears hydrophobic groups grafted at the level ofoxygen atoms originating from the —OH groups and/or the —CH₂OH groupsfixed to the chitosan ring, and having the formula:

in which n, m and R⁴ have the meanings designated above.

According to another embodiment of the invention, in the inclusioncomplex, chitosan bears hydrophobic groups grafted at the level ofoxygen atoms originating from the —OH groups and/or the —CH₂OH groupsfixed to the chitosan ring, and having the formula:

in which

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

in which n, m and R⁴ have the meanings designated above, and providedthat R represents at least one group of formula

In “Biomacromolecules 2002, 3, 1126-1128”, Sashiwa et al. describe the“one-pot” reaction of O-acylation of chitosan. Methyl sulphonate,MeSO₃H, makes it possible to protect the —NH₂ from the N-acylation togive predominantly compounds grafted at the level of the oxygen atoms ofthe chitosan, and more particularly at the level of the primary —OHfunction of the chitosan (—CH₂OH).

The infrared (IR) spectra of the products obtained show a characteristicband of an ester bond at 1700 cm⁻¹. Moreover, the proton NMR spectracompared with that of native chitosan confirm the presence of the alkylchains.

According to another particular embodiment, in the inclusion complex ofthe invention, chitosan bears hydrophobic groups grafted at the level ofcertain oxygen atoms and of certain nitrogen atoms, and has the formula:

in which n, m and R⁴ have the meanings designated above.

According to another particular embodiment, in the inclusion complex ofthe invention, chitosan bears hydrophobic groups grafted at the level ofcertain oxygen atoms and of certain nitrogen atoms, and has the formula:

in which

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

in which n, m and R⁴ have the meanings designated above, and providedthat R represents at least one group of formula

According to yet another embodiment, in the inclusion complex

-   -   the polysaccharide is a dextran bearing hydrophobic groups,        fixed by oxygen atoms of said dextran and representing groups of        formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents dextran,        -   the cyclodextrin is α-CD.

Dextran is a polymer of dextrose.

The IR spectrum of O-palmitoyl-dextran shows an absorption bandcorresponding to the carbonyl of the ester group.

According to another embodiment, in the inclusion complex

-   -   the polysaccharide is hyaluronic acid bearing hydrophobic        groups, fixed by oxygen atoms of said hyaluronic acid and        representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents hyaluronic acid,        -   the cyclodextrin is α-CD.

According to yet another embodiment, in the inclusion complex of theinvention

-   -   the polysaccharide is an amylopectin bearing hydrophobic groups        fixed by oxygen atoms of said amylopectin and representing        groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents hyaluronic acid,        -   the cyclodextrin is α-CD.

Comparison of the IR spectra of O-palmitoyl-amylopectin and nativeamylopectin confirms the presence of the carbonyl of the ester group inthe product obtained.

According to another embodiment, in the inclusion complex of theinvention

-   -   the polysaccharide is a pullulan bearing hydrophobic groups        fixed by oxygen atoms of said pullulan and representing groups        of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents pullulan,        -   the cyclodextrin is α-CD.

Pullulan consists of maltotriose units. The three units of glucose thatmake up maltotriose are joined together by a glycosidic bond of theα-1,4 type, whereas the maltotrioses are joined together by glycosidicbonds of the α-1,6 type. It is grafted, in particular with palmiticacid. Comparison of the IR spectra of O-palmitoyl-pullulan and of nativepullulan confirms the presence of the carbonyl of the ester group in theproduct obtained.

According to another embodiment, in the inclusion complex of theinvention

-   -   the polysaccharide is a heparin bearing hydrophobic groups fixed        by nitrogen atoms of said heparin and representing groups of        formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents heparin,        -   or the polysaccharide is a heparin bearing hydrophobic            groups fixed by oxygen atoms of said heparin, these oxygens            possibly originating from the hydroxyl or carboxyl groups of            the heparin, and representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents heparin,        -   and the cyclodextrin is α-CD.

According to another advantageous aspect, in the inclusion complex ofthe invention

-   -   the polysaccharide is a carrageenan bearing hydrophobic groups        fixed by nitrogen atoms of said carrageenan sulphate and        representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents carrageenan,        -   or the polysaccharide is a carrageenan bearing hydrophobic            groups fixed by oxygen atoms of said carrageenan, these            oxygens possibly originating from the hydroxyl or carboxyl            groups of carrageenan, and representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents carrageenan,        -   and the cyclodextrin is α-CD.

According to another advantageous aspect, in the inclusion complex ofthe invention

-   -   the polysaccharide is a hyaluronic acid bearing hydrophobic        groups fixed by nitrogen atoms of said hyaluronic acid and        representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents hyaluronic acid,        -   or the polysaccharide is a hyaluronic acid bearing            hydrophobic groups fixed by oxygen atoms of said hyaluronic            acid, these oxygens possibly originating from the hydroxyl            or carboxyl groups of the hyaluronic acid, and representing            groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents hyaluronic acid,        -   and the cyclodextrin is α-CD.

According to another advantageous aspect, in the inclusion complex ofthe invention

-   -   the polysaccharide is a glycosaminoglycan bearing hydrophobic        groups fixed by nitrogen atoms of said glycosaminoglycan and        representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents glycosaminoglycan,        -   or the polysaccharide is a glycosaminoglycan bearing            hydrophobic groups fixed by oxygen atoms of said            glycosaminoglycan, these oxygens possibly originating from            the hydroxyl or carboxyl groups of glycosaminoglycan, and            representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents glycosaminoglycan,        -   and the cyclodextrin is α-CD.

According to another advantageous aspect, in the inclusion complex ofthe invention

-   -   the polysaccharide is a carrageenan sulphate bearing hydrophobic        groups fixed by nitrogen atoms of said carrageenan sulphate and        representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents carrageenan sulphate,        -   or the polysaccharide is a carrageenan sulphate bearing            hydrophobic groups fixed by oxygen atoms of said carrageenan            sulphate, these oxygens possibly originating from the            hydroxyl or carboxyl groups of carrageenan sulphate, and            representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents carrageenan sulphate,        -   and the cyclodextrin is α-CD.

According to another advantageous aspect, in the inclusion complex ofthe invention

-   -   the polysaccharide is a dextran sulphate bearing hydrophobic        groups fixed by nitrogen atoms of said dextran sulphate and        representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents dextran sulphate,        -   or the polysaccharide is a dextran sulphate bearing            hydrophobic groups fixed by oxygen atoms of said dextran            sulphate, these oxygens possibly originating from the            hydroxyl or carboxyl groups of dextran sulphate, and            representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents dextran sulphate,        -   and the cyclodextrin is α-CD.

According to another advantageous aspect, in the inclusion complex ofthe invention

-   -   the polysaccharide is a cellulose sulphate bearing hydrophobic        groups fixed by nitrogen atoms of said cellulose sulphate and        representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents cellulose sulphate,        -   or the polysaccharide is a cellulose sulphate bearing            hydrophobic groups fixed by oxygen atoms of said cellulose            sulphate, these oxygens possibly originating from the            hydroxyl or carboxyl groups of cellulose sulphate, and            representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents cellulose sulphate,        -   and the cyclodextrin is α-CD.

According to another advantageous aspect, in the inclusion complex ofthe invention

-   -   the polysaccharide is a heparan sulphate bearing hydrophobic        groups fixed by nitrogen atoms of said heparan sulphate and        representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents heparan sulphate,        -   or the polysaccharide is a heparan sulphate bearing            hydrophobic groups fixed by oxygen atoms of said heparan            sulphate, these oxygens possibly originating from the            hydroxyl or carboxyl groups of the heparan sulphate, and            representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents heparan sulphate,        -   and the cyclodextrin is α-CD.

According to another advantageous aspect, in the inclusion complex ofthe invention

-   -   the polysaccharide is a keratan sulphate bearing hydrophobic        groups fixed by nitrogen atoms of said keratan sulphate and        representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents keratan sulphate,    -   or the polysaccharide is a keratan sulphate bearing hydrophobic        groups fixed by oxygen atoms of said keratan sulphate, these        oxygens possibly originating from the hydroxyl or carboxyl        groups of keratan sulphate, and representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents keratan sulphate,        -   and the cyclodextrin is α-CD.

According to another advantageous aspect, in the inclusion complex ofthe invention

-   -   the polysaccharide is a chondroitin sulphate bearing hydrophobic        groups fixed by nitrogen atoms of said chondroitin sulphate and        representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents chondroitin sulphate,        -   or the polysaccharide is a chondroitin sulphate bearing            hydrophobic groups fixed by oxygen atoms of said chondroitin            sulphate, these oxygens possibly originating from the            hydroxyl or carboxyl groups of chondroitin sulphate, and            representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents chondroitin sulphate,        -   and the cyclodextrin is α-CD.

According to another advantageous aspect, in the inclusion complex ofthe invention

-   -   the polysaccharide is a dextrin sulphate bearing hydrophobic        groups fixed by nitrogen atoms of said dextrin sulphate and        representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents dextrin sulphate,        -   or the polysaccharide is a dextrin sulphate bearing            hydrophobic groups fixed by oxygen atoms of said dextrin            sulphate, these oxygens possibly originating from the            hydroxyl or carboxyl groups of dextrin sulphate, and            representing groups of formula

in which

-   -   R⁴ has the meanings designated above,    -   * represents dextrin sulphate,        -   and the cyclodextrin is α-CD.

According to an advantageous embodiment, the invention also relates to aparticle with a size in the range from 10 nm to 100,000 nm containinginclusion complexes formed by interaction between at least

-   -   a polysaccharide comprising hydrophobic groups bound covalently        to said polysaccharide, optionally functionalized with a ligand        selected from antibodies, antibody fragments, receptors, lectins        or biotin or derivatives thereof,    -   a cyclodextrin in the form of monomer, optionally functionalized        with a ligand selected from antibodies, antibody fragments,        receptors, lectins or biotin or derivatives thereof.

The invention also relates to a particle of a size in the range from 50nm to 10,000 nm containing inclusion complexes between:

-   -   a polysaccharide comprising hydrophobic groups bound covalently        to said polysaccharide, optionally functionalized with a ligand        selected from antibodies, antibody fragments, receptors, lectins        or biotin or derivatives thereof,    -   and a cyclodextrin in the form of monomer, optionally        functionalized with a ligand selected from antibodies, antibody        fragments, receptors, lectins or biotin or derivatives thereof.

According to an advantageous embodiment, the invention relates tonanometric particles, of a size in the range from 10 nm to 1000 nm andmicrometric particles of a size in the range from 1000 nm to 100,000 nm.

The invention relates to nanometric particles, of a size in the rangefrom 50 nm to 1000 nm and micrometric particles of a size in the rangefrom 1000 nm to 10,000 nm.

According to an advantageous aspect, the invention relates to a particleof a size in the range from 10 nm to 100,000 nm containing inclusioncomplexes according to the invention formed by interaction between atleast:

-   -   one polysaccharide selected from chitosan, dextran, hyaluronic        acid, amylose, amylopectin, pullulan, heparin, chitin, cellulose        derivatives, heparan sulphate, dermatan sulphate, keratan        sulphate, chondroitin sulphate, cellulose sulphate, dextran        sulphate, dextrin sulphate, starch, pectin, the alginates, the        carrageenans, fucan, curdlan, xylan, polyguluronic acid,        xanthan, arabinan, polymannuronic acid, and derivatives thereof,        comprising hydrophobic groups selected from linear or branched        alkyl groups or linear or branched alkenyl groups bearing from 1        to 4 C═C double bonds, conjugated or not, said hydrophobic        groups being bound covalently to said polysaccharide, optionally        functionalized with a ligand selected from antibodies, antibody        fragments, receptors, lectins or biotin or derivatives thereof,    -   and one α-cyclodextrin in the form of monomer, optionally        functionalized with a ligand selected from antibodies, antibody        fragments, receptors, lectins or biotin or derivatives thereof.

The particle sizes formed were evaluated by quasi-elastic lightscattering (QELS) on the one hand and by transmission electronmicroscopy (TEM) on the other hand.

Size plays a very important role in connection with the quantity ofactive ingredient encapsulated, the applications envisaged, and theroute of administration of the active ingredient.

The invention also relates to a particle of a size in the range from 10nm to 100,000 nm containing inclusion complexes formed by interactionbetween at least:

-   -   one polysaccharide selected from chitosan, dextran, hyaluronic        acid, amylose, amylopectin, pullulan, heparin, chitin, cellulose        derivatives, heparan sulphate, dermatan sulphate, keratan        sulphate, chondroitin sulphate, cellulose sulphate, dextran        sulphate, dextrin sulphate, starch, pectin, the alginates, the        carrageenans, fucan, curdlan, xylan, polyguluronic acid,        xanthan, arabinan, polymannuronic acid, and derivatives thereof,        comprising hydrophobic groups selected from linear or branched        alkyl groups containing from 2 to 1000 carbon atoms, or linear        or branched, in particular linear, alkenyl groups, which may        contain at least one C═C double bond, said hydrophobic groups        being bound covalently to said polysaccharide, optionally        functionalized with a ligand selected from antibodies, antibody        fragments, receptors, lectins or biotin or derivatives thereof,    -   and one α-cyclodextrin in the form of monomer, optionally        functionalized with a ligand selected from antibodies, antibody        fragments, receptors, lectins or biotin or derivatives thereof.

According to a particular embodiment, the invention also relates toparticles of a size in the range from 10 nm to 100,000 nm containinginclusion complexes formed by interaction between:

-   -   a mixture of at least two polysaccharides selected from        chitosan, dextran, hyaluronic acid, amylose, amylopectin,        pullulan, heparin, chitin, cellulose derivatives, heparan        sulphate, dermatan sulphate, keratan sulphate, chondroitin        sulphate, cellulose sulphate, dextran sulphate, dextrin        sulphate, starch, pectin, the alginates, the carrageenans,        fucan, curdlan, xylan, polyguluronic acid, xanthan, arabinan,        polymannuronic acid, and derivatives thereof, comprising        hydrophobic groups selected from linear or branched alkyl groups        containing from 2 to 1000 carbon atoms, or linear or branched,        in particular linear, alkenyl groups, which may contain at least        one C═C double bond, said hydrophobic groups being bound        covalently to said polysaccharide, optionally functionalized        with a ligand selected from antibodies, antibody fragments,        receptors, lectins or biotin or derivatives thereof,    -   and at least one α-cyclodextrin in the form of monomer,        optionally functionalized with a ligand selected from        antibodies, antibody fragments, receptors, lectins or biotin or        derivatives thereof.

The invention also relates to an encapsulation system containing one ormore particles defined above, and a substance used for its properties inthe pharmaceutical, paramedical, medical device, animal-feed,agrochemical, medical, cosmetic, veterinary, agri-food, pesticide,cosmetotextile, perfumery, and environmental fields (water purificationfor example) or in the paint, building and/or car industry.

A medical device is an instrument, apparatus, equipment or softwareintended to be used for humans or animals for the purposes of:diagnosis, prevention, control, treatment or attenuation of a disease,diagnosis, control, treatment, attenuation or compensation of a wound orof a handicap, investigation or replacement or modification of theanatomy or of a physiological process, or control of conception.

According to an advantageous aspect, the substance has pharmaceuticalproperties and is selected from inorganic compounds and organiccompounds, synthetic or natural.

The encapsulation system defined above according to the invention may beused for preparing suitable compositions in the pharmaceutical, medical,paramedical, medical device, animal-feed, cosmetic, veterinary,agri-food, pesticide, cosmetotextile, perfumery, and environmentalfields (water purification for example) or in the paint, packaging,building and/or car industry.

The substance encapsulated may have pharmaceutical properties and may bean active ingredient for therapeutic use. It may belong to the followinglist, the latter in no case being limiting, to the group of compoundswith vitamin properties, in particular vitamin A, vitamin E, vitamin C,the K vitamins, the B vitamins, vitamin D, antitumour compounds, inparticular paclitaxel, docetaxel, tamoxifen, doxorubicin, analgesics, inparticular paracetamol, anti-inflammatories, in particular diclofenac,ibuprofen, ketoprofen, antibiotics, in particular the penicillins, thetetracyclines, antifungals, in particular ketoconazole, clotrimazole,nystatin, chlorhexidine and derivatives thereof, antiparasitic agents,in particular albendazole, metronidazole, enzymatic compounds, inparticular alkaline phosphatase, acetylcholinesterase, alcoholdehydrogenase, hormonal compounds, in particular testosterone,levonorgestrel, anxiolytics, in particular the benzodiazepines,antidiabetic agents, in particular glicazide, anti-hypertensives, inparticular nifedipine, or vaccines, antivirals, in particular AZT,analgesics or combinations of analgesics, in particular paracetamol,antiepileptics in particular the barbiturates and derivatives, local andgeneral anaesthetics, in particular atropine, but also hypnotics,sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics,in particular antagonists, nerve blocking agents, anticholinergics,cholinomimetics, antimuscarinics, muscarinics, in particularanti-adrenergics, antiarrhythmics, antiarthritics, antiasthmatics,anticonvulsives, antihistamines, anti-nausea agents, antineoplastics,antipyretics, antipruritics, antispasmodics, diuretics, vasodilators,central nervous system stimulants, in particular preparations againstcoughs and colds, decongestants, bone growth stimulants, inhibitors ofbone resorption, immunosuppressants, muscle relaxants, psychostimulants,sedatives, tranquillizers, proteins, peptides or fragments thereof, saidproteins, peptides or fragments being natural, recombinant or producedchemically, nucleic acids (ribonucleotides or deoxyribonucleotides), inparticular single- and double-stranded molecules, gene constructs,expression vectors, antisense molecules and others of the same kind

This substance for therapeutic use may be used in humans and in animals.

The substance encapsulated may also have cosmetic properties and belongto the group of compounds with anti-inflammatory, anti-ageing,anti-ultraviolet (anti-UV), depigmenting, wound-healing, hydrating,perfuming, deodorizing, antibacterial, antiperspirant, cleaning,colouring, and preserving properties.

In a particular encapsulation system, the substance has nutritionalproperties and belongs to the group of compounds with vitamin,enzymatic, and sweetening properties. It may also be an essential oil, acolorant, a preservative, an antioxidant, or a probiotic.

Some molecules or families of molecules that may be encapsulated are:molsidomine, ketoconazole, gliclazide, diclofenac, levonorgestrel,paclitaxel, docetaxel, tamoxifen, hydrocortisone, pancratistatin,ketoprofen, diazepam, ibuprofen, nifedipine, testosterone, tamoxifen,furosemide, tolbutamide, chloramphenicol, the benzodiazepines, naproxen,dexamethasone, diflunisal, anadamide, pilocarpine, daunorubicin,doxorubicin, the essential oils, the terpenes, the terpenoids.

To improve the solubility of the molecules of interest, the use of asolvent or a mixture of solvents may be envisaged, in particular: alkylacetate (ethyl acetate, butyl acetate, methyl acetate), acetone,acetonitrile, acetic acid, methanoic acid, ammonia, acetic anhydride,aniline, anisole, benzene, butanol, butanone, chlorobenzene, chloroform,cyclohexane, cyclopentane, dichloroethane, dichloromethane, diisopropylether, dimethylformamide, dimethylsulphoxide, dioxane, water, ethanol,glycol ether, diethyl ether, ethylene glycol, heptane,hexamethylphosphoramide, hexane, methanol, methyl ethyl ketone,nitrobenzene, pentane, perchloroethylene, propanol, propoxypropane,pyridine, carbon disulphide, tetrachloroethane, tetrahydrofuran,toluene, trichloroethane, trichloroethylene, tremethylpentane, xylene.The fields of use of the compositions containing the encapsulationsystem with the active ingredient are therefore the medical, veterinary,cosmetic, and cosmetotextile fields, the environmental field inparticular connected with water purification, the paint, building or carsector, and perfumery.

The invention also relates to the use of the particles defined above.

The invention thus relates to the use of said particles as medicaments,in particular as adjuvants for vaccination, treatment of burns, and/orfor wound-healing.

The invention also relates to the use of said particles in thepreparation of medicaments having at least one activity for preventing,inhibiting and/or treating fungal, bacterial, viral and/or parasiticinfections on biotic or abiotic surfaces.

The invention also relates to the use of said particles as veterinarymedicaments, in particular as adjuvants for vaccination.

The invention also relates to the use of the particles as a cosmeticagent, in particular as anti-ageing, depigmenting, wound-healing,hydrating, perfuming, deodorizing, antiperspirant, cleaning, colouring,or preserving agent.

The invention also relates to the use of the particles for carrying outa method for preparing devices, in particular wound-healing dressings,said devices comprising said particles, and being able to release saidparticles or one or more active substance(s) of interest contained insaid particles.

The invention also relates to a pharmaceutical composition containing,as active substance, the substance encapsulated in the particles, insolid form, or in the form of solution or suspension in a physiologicalmedium, optionally enriched with an excipient such as glucose, sucroseor any other pharmaceutically acceptable excipient, usable for thefollowing routes:

parenteral, oral, cutaneous, subcutaneous, nasal, pulmonary or ocular,and for any administration at the level of a mucous membrane, or at thelevel of a precise site (tumour, lumen of certain blood vessels),in the form of pills (tablets), soft capsules, hard capsules (gelatincapsules), powders, granules, soluble or dispersible tablets, patches,implants, suppositories, solutions, suspension, syrup, pastes, creams,gels, emulsions, sprays, lotions, ointments, shampoos.

The invention also relates to a pharmaceutical composition containing,as active substance, a substance encapsulated in inclusion complexes orin particles defined above, together with a pharmaceutically acceptablevehicle, in solid form, or in the form of solution or suspension in aphysiological medium, usable by the following routes:

parenteral, oral, cutaneous, subcutaneous, nasal, pulmonary or ocular,and for any administration at the level of a mucous membrane, inparticular in the form of pills (tablets), soft capsules, hard capsules(gelatin capsules), powders, granules, soluble or dispersible tablets,patches, implants, suppositories, solutions, suspension, syrup, pastes,creams, gels, emulsions, sprays, lotions, or ointments.

The forms that are advantageous in the pharmaceutical field are tablets,soft capsules, hard capsules (gelatin capsules), powders, granules,patches, implants, suppositories, solutions, suspensions, syrups,pastes, creams, gels, emulsions, sprays, lotions and ointments.

The forms that are advantageous in the paramedical field are dressings,catheters, compresses, gauze, hydrophilic cotton, normal saline, sprayetc.

The forms that are advantageous in the field of medical devices areimplants, prostheses, equipment for washing and disinfectinginstruments, compresses, dressings (in particular wound-healingdressings), sprays, gauzes, hydrophilic cotton etc.

The forms that are advantageous in the veterinary field are the oralforms (tablets, powders, soft capsules, hard capsules (gelatincapsules), granules, pastes, solutions, suspensions), injectable forms(solutions, suspensions) and topical forms the action of which may belocal or systemic (sprays, collars, ear tags, powders, lotions,ointments, shampoos, patches, emulsions, milk, gel, cream).

The forms that are advantageous in the food sector are solutions,emulsions, pastes, gels, powders used alone or incorporated in foodpreparations; in the field of food supplements: mainly the oral forms(powders, tablets, hard capsules (gelatin capsules), granules, softcapsules, pastes, solutions, suspensions, infusions).

The invention also relates to a cosmetic composition containing, asactive substance, the substance encapsulated in the particles, andcontaining cosmetically acceptable excipients, usable in the form ofgels, pastes, ointments, lotions, creams, milks, sticks, shampoos,powders, aerosols, and patches.

The invention also relates to a method for preparing an inclusioncomplex comprising a step of mixing:

-   -   a polysaccharide comprising hydrophobic groups bound covalently        by a nitrogen atom or by one or more oxygen atoms to said        polysaccharide,    -   and a cyclodextrin (CD) in the form of monomer, to obtain an        inclusion complex in which said polysaccharide and said        cyclodextrin are bound non-covalently.

According to a particular embodiment, the method for the preparation,according to the invention, of an inclusion complex comprises a step ofmixing:

-   -   a polysaccharide comprising hydrophobic groups bound covalently        to the polysaccharide by a nitrogen atom of said polysaccharide,    -   and a cyclodextrin (CD) in the form of monomer, to obtain an        inclusion complex in which said polysaccharide and said        cyclodextrin are bound non-covalently.

According to a particular embodiment, the method for the preparation,according to the invention, of an inclusion complex comprises a step ofmixing at least:

-   -   one polysaccharide comprising hydrophobic groups bound        covalently to the polysaccharide by a nitrogen atom of said        polysaccharide, and    -   one cyclodextrin (CD) in the form of monomer, to obtain an        inclusion complex in which said polysaccharide and said        cyclodextrin are bound non-covalently.

The invention also relates to a method for preparing an inclusioncomplex as defined above, comprising a step of mixing at least

-   -   one polysaccharide in the form of suspension in a solvent, in        particular water comprising hydrophobic groups bound covalently        to the polysaccharide by oxygen atoms of said polysaccharide,        and    -   one α-cyclodextrin (CD) in the form of monomer,

to obtain an inclusion complex in which said polysaccharide and saidcyclodextrin are bound non-covalently,

and in particular comprising a step of mixing:

-   -   a polysaccharide selected from chitosan, dextran, hyaluronic        acid, amylose, amylopectin, pullulan, heparin, chitin, heparan        sulphate, dermatan sulphate, keratan sulphate, chondroitin        sulphate, cellulose sulphate, dextran sulphate, dextrin        sulphate, starch, pectin, the alginates, the carrageenans,        fucan, curdlan, xylan, polyguluronic acid, xanthan, arabinan,        polymannuronic acid, and derivatives thereof,

said polysaccharide comprising hydrophobic groups of formula:

in which

-   -   * represents the polysaccharide,    -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and bearing at least one C═C double bond, in            particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,    -   and an α-cyclodextrin (CD) having the formula:

in which

-   -   p is equal to 6,    -   R¹, R² and R³, which may be identical or different, in        particular identical, are hydrogen atoms, alkyl groups        comprising 1 to 3 carbon atoms, selected from methyls, ethyls,        propyls, isopropyls, —NH₂ amino groups, —NH₃ ⁺ ammonium groups,        or —SO₄ ²⁻ sulphate groups and are in particular hydrogen atoms        or methyl groups,        said CD being alpha-cyclodextrin (α-CD) in the form of monomer,        to obtain an inclusion complex in which said polysaccharide and        said cyclodextrin are bound non-covalently.

In another aspect, the invention also relates to a method for preparingan inclusion complex as defined above, comprising a step of mixing atleast:

-   -   a polysaccharide comprising hydrophobic groups bound covalently        to the polysaccharide by oxygen atoms of said polysaccharide,        and    -   an α-cyclodextrin (CD) in the form of monomer in suspension in a        solvent, in particular water,        to obtain an inclusion complex in which said polysaccharide and        said cyclodextrin are bound non-covalently,        and in particular comprising a step of mixing:    -   a polysaccharide selected from chitosan, dextran, hyaluronic        acid, amylose, amylopectin, pullulan, heparin, chitin, heparan        sulphate, dermatan sulphate, keratan sulphate, chondroitin        sulphate, cellulose sulphate, dextran sulphate, dextrin        sulphate, starch, pectin, the alginates, the carrageenans,        fucan, curdlan, xylan, polyguluronic acid, xanthan, arabinan,        polymannuronic acid, and derivatives thereof,        said polysaccharide comprising hydrophobic groups of formula:

in which

-   -   * represents the polysaccharide,    -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and bearing at least one C═C double bond, in            particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups, and    -   an α-cyclodextrin (CD) having the formula:

in which

-   -   p is equal to 6,    -   R¹, R² and R³, which may be identical or different, in        particular identical, are hydrogen atoms, alkyl groups        comprising 1 to 3 carbon atoms, selected from methyls, ethyls,        propyls, isopropyls, —NH₂ amino groups, —NH₃ ⁺ ammonium groups,        or —SO₄ ²⁻ sulphate groups, and are in particular hydrogen atoms        or methyl groups,        said CD being alpha-cyclodextrin (α-CD) in the form of monomer,        to obtain an inclusion complex in which said polysaccharide and        said cyclodextrin are bound non-covalently.

In yet another particular embodiment, the invention also relates to amethod for preparing an inclusion complex as defined above, comprising astep of mixing at least:

-   -   one polysaccharide in the form of suspension in a solvent, in        particular water comprising hydrophobic groups bound covalently        to the polysaccharide by oxygen atoms of said polysaccharide,    -   and one α-cyclodextrin (CD) in the form of monomer, preferably        in suspension in a solvent, in particular water,        to obtain an inclusion complex in which said polysaccharide and        said cyclodextrin are bound non-covalently,        and in particular comprising a step of mixing:    -   a polysaccharide selected from chitosan, dextran, hyaluronic        acid, amylose, amylopectin, pullulan, heparin, chitin, heparan        sulphate, dermatan sulphate, keratan sulphate, chondroitin        sulphate, cellulose sulphate, dextran sulphate, dextrin        sulphate, starch, pectin, the alginates, the carrageenans,        fucan, curdlan, xylan, polyguluronic acid, xanthan, arabinan,        polymannuronic acid, and derivatives thereof,        said polysaccharide comprising hydrophobic groups of formula:

in which

-   -   * represents the polysaccharide,    -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and bearing at least one C═C double bond, in            particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups, and    -   an α-cyclodextrin (CD) having the formula:

in which

-   -   p is equal to 6,    -   R¹, R² and R³, which may be identical or different, in        particular identical, are hydrogen atoms, alkyl groups        comprising 1 to 3 carbon atoms, selected from methyls, ethyls,        propyls, isopropyls, —NH₂ amino groups, —NH₃ ⁺ ammonium groups,        or —SO₄ ²⁻ sulphate groups, and are in particular hydrogen atoms        or methyl groups,        said CD being alpha-cyclodextrin (α-CD) in the form of monomer,        to obtain an inclusion complex in which said polysaccharide and        said cyclodextrin are bound non-covalently.

According to another embodiment, the method for preparing an inclusioncomplex comprises a step of mixing

-   -   a polysaccharide comprising hydrophobic groups bound covalently        to the polysaccharide by oxygen atoms of said polysaccharide,    -   and a cyclodextrin (CD) in the form of monomer,        to obtain an inclusion complex in which said polysaccharide and        said cyclodextrin are bound non-covalently.

According to another advantageous embodiment, the method for preparingan inclusion complex comprises a step of mixing at least:

-   -   one polysaccharide comprising hydrophobic groups bound        covalently to the polysaccharide by oxygen atoms of said        polysaccharide,    -   and one cyclodextrin (CD) in the form of monomer,        to obtain an inclusion complex in which said polysaccharide and        said cyclodextrin are bound non-covalently.

The invention also relates to a method for preparing an inclusioncomplex comprising a step of mixing:

-   -   a chitosan comprising hydrophobic groups fixed covalently to the        chitosan by one or more oxygen atoms of said chitosan,    -   and an α-cyclodextrin (CD),        and in particular comprising a step of mixing between a chitosan        of formula I:

or a chitosan of formula II:

in which

-   -   m represents the number of D-glucosamine units,    -   n represents the number of N-acetyl-D-glucosamine units,        provided that the degree of deacetylation representing the        percentage of m relative to the total number of units is greater        than 50%,    -   R⁴ represents a hydrophobic group and is selected from:        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and bearing from 1 to 4 C═C double bonds,            conjugated or not, in particular the            —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or —(CH₂)₇—CH═CH—(CH₂)₇—CH₃            groups,

and an α-cyclodextrin (CD) of formula:

in which

-   -   p is equal to 6,    -   R¹, R² and R³, which may be identical or different, in        particular identical, are hydrogen atoms, alkyl groups        comprising 1 to 3 carbon atoms, selected from methyls, ethyls,        propyls, isopropyls, —NH₂ amino groups, —NH₃ ⁺ ammonium groups,        or —SO₄ ⁻ sulphate groups, and are in particular hydrogen atoms        or methyl groups,        said CD being alpha-cyclodextrin (α-CD) in the form of monomer,        at a concentration in the range from 0.01 to 9000 g/L of aqueous        medium, in particular in the range from 1 to 300 g/L of aqueous        medium, and in particular equal to approximately 200 g/L of        aqueous medium,        to obtain an inclusion complex in which the chitosan of formula        I and the cyclodextrin are bound non-covalently.

According to a particular embodiment, the method for preparing aninclusion complex comprises a step of mixing:

-   -   several polysaccharides comprising hydrophobic groups fixed        covalently to the aforesaid polysaccharides by a nitrogen atom        and/or by one or more oxygen atoms of said polysaccharides, the        polysaccharides being chitosan, dextran, hyaluronic acid,        amylose, amylopectin, pullulan, heparin, chitin, cellulose        derivatives, heparan sulphate, dermatan sulphate, keratan        sulphate, chondroitin sulphate, cellulose sulphate, dextran        sulphate, dextrin sulphate, starch, pectin, the alginates, the        carrageenans, fucan, curdlan, xylan, polyguluronic acid,        xanthan, arabinan, polymannuronic acid, and derivatives thereof,    -   a cyclodextrin (CD),        to obtain an inclusion complex in which the polysaccharides and        the cyclodextrin are bound non-covalently. The use of several        polysaccharides may offer the advantage of modulating the        properties of the particles by altering the ratio of the        polysaccharides. Thus, it is expected that the size, the overall        charge and the intended applications of these particles may be        modulated.

According to a particular embodiment, the method for preparing aninclusion complex comprises a step of mixing:

-   -   a polysaccharide selected from chitosan, dextran, hyaluronic        acid, amylose, amylopectin, pullulan, heparin, chitin, cellulose        derivatives, heparan sulphate, dermatan sulphate, keratan        sulphate, chondroitin sulphate, cellulose sulphate, dextran        sulphate, dextrin sulphate, starch, pectin, the alginates, the        carrageenans, fucan, curdlan, xylan, polyguluronic acid,        xanthan, arabinan, polymannuronic acid, and derivatives thereof,        and is in particular chitosan,        said polysaccharide comprising hydrophobic groups of formula:

in which

-   -   * represents the polysaccharide,    -   R⁴ represents:        -   a linear or branched alkyl group containing from 1 to 20            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 20 carbon            atoms and bearing from 1 to 4 C═C double bonds, conjugated            or not, in particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups, and    -   a cyclodextrin (CD) having the formula:

in which

-   -   p is an integer equal to 6,    -   R¹, R² and R³, which may be identical or different, in        particular identical, are hydrogen atoms, alkyl groups        comprising 1 to 3 carbon atoms, selected from methyls, ethyls,        propyls, isopropyls, —NH₂ amino groups, —NH₃ ⁺ ammonium groups,        or —SO₄ ²⁻ sulphate groups, and are in particular hydrogen atoms        or methyl groups,        said CD being alpha-cyclodextrin (α-CD) in the form of monomer,        to obtain an inclusion complex in which said polysaccharide and        said cyclodextrin are bound non-covalently.

According to a particular embodiment, the method for preparing aninclusion complex comprises a step of mixing at least

-   -   one polysaccharide selected from chitosan, dextran, hyaluronic        acid, amylose, amylopectin, pullulan, heparin, chitin, cellulose        derivatives, heparan sulphate, dermatan sulphate, keratan        sulphate, chondroitin sulphate, cellulose sulphate, dextran        sulphate, dextrin sulphate, starch, pectin, the alginates, the        carrageenans, fucan, curdlan, xylan, polyguluronic acid,        xanthan, arabinan, polymannuronic acid, and derivatives thereof,        and is in particular chitosan,        said polysaccharide comprising hydrophobic groups of formula:

in which

-   -   * represents the polysaccharide,    -   R⁴ represents:        -   a linear or branched alkyl group containing from 1 to 20            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 20 carbon            atoms and bearing from 1 to 4 C═C double bonds, conjugated            or not, in particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,    -   and a cyclodextrin (CD) having the formula:

in which

-   -   p is an integer equal to 6,    -   R¹, R² and R³, which may be identical or different, in        particular identical, are hydrogen atoms, alkyl groups        comprising 1 to 3 carbon atoms, selected from methyls, ethyls,        propyls, isopropyls, —NH₂ amino groups, —NH₃ ⁺ ammonium groups,        or —SO₄ ²⁻ sulphate groups, and are in particular hydrogen atoms        or methyl groups,        said CD being alpha-cyclodextrin (α-CD) in the form of monomer,        to obtain an inclusion complex in which said polysaccharide and        said cyclodextrin are bound non-covalently.

According to a particular aspect, the method for preparing an inclusioncomplex comprises a step of mixing the polysaccharide:

-   -   in aqueous solution at a concentration in the range from 0.01 to        9000 g/L, in particular in the range from 1 to 600 g/L, and in        particular equal to approximately 10 g/L, the aqueous solvent        being selected from pure water, an aqueous solution with pH in        the range from 1 to 7 or from 7 to 14, in particular in the        range from 5 to 7, or a physiological serum solution optionally        enriched with glucose or containing any other excipient for        pharmaceutical, medical, paramedical, medical-device,        animal-feed, cosmetic, veterinary, agri-food, pesticide,        cosmetotextile, perfumery, and environmental use (water        purification for example) or in the paint, packaging, building        and/or car industry,    -   or in dispersion in an aqueous medium selected from pure water,        an aqueous solution with pH in the range from 1 to 7 or from 7        to 14, in particular in the range from 5 to 7, or a        physiological serum solution optionally enriched with glucose or        containing any other excipient for pharmaceutical, medical,        paramedical, medical-device, animal-feed, cosmetic, veterinary,        agri-food, pesticide, cosmetotextile, perfumery, and        environmental use (water purification for example) or in the        paint, packaging, building and/or car industry, with a        cyclodextrin, α-CD.

The method for preparing an inclusion complex comprises a step of mixingthe polysaccharide

-   -   in aqueous solution at a concentration in the range from 1 to        150 g/L, in particular in the range from 5 to 50 g/L, and in        particular equal to approximately 10 g/L, the aqueous solvent        being selected from pure water, an aqueous solution with pH in        the range from 1 to 7 or from 7 to 12, in particular in the        range from 5 to 7, or a physiological serum solution optionally        enriched with glucose or containing any other excipient for        pharmaceutical, cosmetic, agri-food or veterinary use,    -   or in dispersion in an aqueous medium selected from pure water,        an aqueous solution with pH in the range from 1 to 7 or from 7        to 12, in particular in the range from 5 to 7, or a        physiological serum solution optionally enriched with glucose or        containing any other excipient for pharmaceutical, cosmetic,        agri-food or veterinary use, with a cyclodextrin, α-CD.

In the method for preparing an inclusion complex, the step of mixing thepolysaccharide in aqueous solution or dispersion is carried out at aconcentration in the range from 0.01 to 9000 g/L, in particular in therange from 1 to 600 g/L, and in particular equal to approximately 10g/L,

with a cyclodextrin, in particular α-CD, at a concentration in the rangefrom 0.01 to 9000 g/L, in particular in the range from 1 to 300 g/L, andin particular equal to approximately 200 g/L.

In the method for preparing an inclusion complex, the step of mixing thepolysaccharide in aqueous solution or dispersion is carried out at aconcentration in the range from 1 to 150 g/L, in particular in the rangefrom 5 to 50 g/L, and in particular equal to approximately 10 g/L,

with a cyclodextrin, α-CD, at a concentration in the range from 1 to 150g/L, in particular in the range from 5 to 50 g/L, and in particularequal to approximately 10 g/L.

According to another embodiment, the method for preparing an inclusioncomplex comprises a step of mixing the polysaccharide in dispersion inan aqueous medium, the concentration of said polysaccharide being in therange from 0.01 to 9000 g/L of aqueous medium, in particular being inthe range from 1 to 600 g/L of aqueous medium, and in particular beingequal to approximately 1 g/L or equal to approximately 10 g/L of aqueousmedium,

with a cyclodextrin, α-CD, at a concentration in the range from 0.01 to9000 g/L of aqueous medium, in particular in the range from 0.01 to 300g/L of aqueous medium, and in particular equal to approximately 10 g/Lof aqueous medium.

According to another embodiment, the method for preparing an inclusioncomplex comprises a step of mixing the polysaccharide in dispersion inan aqueous medium, the weight of said polysaccharide being in the rangefrom 1 to 150 g/L of aqueous medium, in particular being in the rangefrom 1 to 10 g/L of aqueous medium, and in particular being equal toapproximately 1 g/L or equal to approximately 10 g/L of aqueous medium,with a cyclodextrin, α-CD, at a concentration in the range from 1 to 150g/L of aqueous medium, in particular in the range from 1 to 50 g/L ofaqueous medium, and in particular equal to approximately 10 g/L ofaqueous medium.

The complexes also form when the polysaccharide is used in the form of asuspension.

The method for preparing an inclusion complex comprises a step of mixingthe polysaccharide with a cyclodextrin, α-CD,

-   -   the percentage by weight of said polysaccharide being in the        range from 0.01% to 90%, and in particular from 0.5% to 50%,    -   the percentage by weight of the cyclodextrin being in the range        from 0.01% to 90%, and in particular from 0.5% to 50%,    -   the percentage by weight of water or of aqueous medium being in        the range from 10% to 99.99%, and in particular from 50% to        99.5%.

The method for preparing an inclusion complex comprises a step of mixingthe polysaccharide with a cyclodextrin, α-CD,

-   -   the percentage by weight of said polysaccharide being in the        range from 0.1% to 15%, and in particular from 0.5% to 5%,    -   the percentage by weight of the cyclodextrin being in the range        from 0.1% to 15%, and in particular from 0.5% to 5%,    -   the percentage by weight of water or of aqueous medium being in        the range from 70% to 99.9%, and in particular from 90% to 99%.

According to a particular embodiment of the method for preparing aninclusion complex, the step of mixing the polysaccharide with acyclodextrin, α-CD, is carried out with stirring, in particularmagnetic, at a stirring speed in the range from 10 to 1000 rpm, inparticular at a stirring speed in the range from 100 to 500 rpm, and inparticular at a stirring speed equal to approximately 200 rpm,

at a temperature in the range from 1 to 100° C., in particular at atemperature in the range from 10 to 35° C., and in particular at atemperature equal to approximately 20° C., the duration of stirringbeing in the range from 6 hours to 15 days, in particular from 24 hoursto 96 hours, and in particular equal to approximately 72 hours.

According to a particular embodiment of the method for preparing aninclusion complex, the step of mixing the polysaccharide with acyclodextrin, α-CD,

is carried out with stirring, in particular magnetic, at a stirringspeed in the range from 50 to 1000 rpm, in particular at a stirringspeed in the range from 100 to 500 rpm, and in particular at a stirringspeed equal to approximately 200 rpm,at a temperature in the range from 4 to 60° C., in particular at atemperature in the range from 10 to 35° C., and in particular at atemperature equal to approximately 20° C.,the duration of stirring being in the range from 24 hours to 15 days, inparticular in the range from 24 hours to 48 hours, and in particularequal to approximately 36 hours.

The method for preparing an inclusion complex also comprises a step ofpreparing a polysaccharide bearing hydrophobic groups, by a reaction ofN-acylation

-   -   between the polysaccharide and an acid chloride of formula        R⁴—C(O)C1, in which

R⁴ represents:

-   -   a linear or branched alkyl group containing from 1 to 1000        carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃        groups,    -   a linear or branched alkenyl group containing 2 to 1000 carbon        atoms and bearing at least one C═C double bond, in particular        the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or —(CH₂)₇—CH═CH—(CH₂)₇—CH₃        groups,    -   or between the polysaccharide and a fatty acid of formula        R⁴—CO₂H, in which R⁴ has the meanings designated above,        in the presence of a coupling agent such as the        N-(3-dimethylaminopropyl)-N-ethylcarbodiimide chloride (EDCI) at        ambient temperature for 24 hours,    -   or between the polysaccharide and a cyclic acid anhydride of        formula R⁴—CO—O—CO—R⁴, in which R⁴ has the meanings designated        above,        to give a polysaccharide bearing hydrophobic groups fixed        covalently to the polysaccharide by a nitrogen atom of said        polysaccharide.

The method for preparing an inclusion complex also comprises a step ofpreparing a polysaccharide bearing hydrophobic groups, by a reaction ofN-acylation

-   -   between the polysaccharide and an acid chloride of formula        R⁴—C(O)Cl,        in which

R⁴ represents:

-   -   a linear or branched alkyl group containing from 1 to 20 carbon        atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃ groups,    -   a linear or branched alkenyl group containing 2 to 20 carbon        atoms and bearing from 1 to 4 C═C double bonds, conjugated or        not, in particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or        —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,    -   or between the polysaccharide and a fatty acid of formula        R⁴—CO₂H, in which R⁴ has the meanings designated above,        in the presence of a coupling agent such as        N-(3-dimethylaminopropyl)-N-ethylcarbodiimide chloride (EDCI) at        ambient temperature for 24 hours,    -   or between the polysaccharide and a cyclic acid anhydride of        formula R⁴—CO—O—CO—R⁴, in which R⁴ has the meanings designated        above,        to give a polysaccharide bearing hydrophobic groups fixed        covalently to the polysaccharide by a nitrogen atom of said        polysaccharide.

The reaction of N-acylation relates in particular to chitosan. It iscarried out by the action of an acid chloride (Li, Y-Y. et al., 2006, J.Appi. Polym. Sci. 102, 1968-1973), of a carboxylic acid, less reactivethan the acid chloride, in the presence of the coupling agent EDCI, orby the action of acid anhydrides (Lee, K. Y. et al., 1995, Biomaterials16, 1211-1216; Lee, M. Y. et al., 2005, Int. J. Biol. Macromol., 36,152-158; Mourya, V. K. and Inamdar, N. N. (2008). React. Funct. Polym.68 (6), 1013-1051).

Another embodiment of the method for preparing an inclusion complexcomprises a step of preparing a polysaccharide bearing hydrophobicgroups, by a reaction of O-acylation between said polysaccharidedissolved in methanesulphonate and 1 to 20 equivalents per unit ofpolysaccharide, of fatty acid chloride, the reaction being carried outat ambient temperature,

said polysaccharide and acid chloride being as designated above,to give a polysaccharide bearing hydrophobic groups fixed covalently tothe polysaccharide by one or more oxygen atoms of said polysaccharide.

Another embodiment of the method for preparing an inclusion complexcomprises a step of mixing at least:

-   -   one chitosan comprising hydrophobic groups fixed covalently to        the chitosan by a nitrogen atom and/or by one or more oxygen        atoms of said chitosan,    -   and one cyclodextrin (CD),        to obtain an inclusion complex in which the chitosan and the        cyclodextrin are bound non-covalently.

Another embodiment of the method for preparing an inclusion complexcomprises a step of mixing at least:

-   -   one chitosan comprising hydrophobic groups fixed covalently to        the chitosan by a nitrogen atom and/or by one or more oxygen        atoms of said chitosan,    -   and one cyclodextrin (CD),        to obtain an inclusion complex in which the chitosan and the        cyclodextrin are bound non-covalently.

According to a particular embodiment, the method for preparing aninclusion complex comprises a step of mixing between

-   -   a chitosan of formula:

or a chitosan of formula:

in which

-   -   m represents the number of deacetylated units,    -   n represents the number of acetylated units,        provided that the degree of deacetylation representing the        percentage of m relative to the total number of units is greater        than 50%,    -   R⁴ represents a hydrophobic group and is selected from:        -   a linear or branched alkyl group containing from 1 to 20            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 20 carbon            atoms and bearing from 1 to 4 C═C double bonds, conjugated            or not, in particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,    -   and a cyclodextrin (CD) of formula:

in which

-   -   p is an integer equal to 6,    -   R¹, R² and R³, which may be identical or different, in        particular identical, are hydrogen atoms, alkyl groups        comprising 1 to 3 carbon atoms, selected from methyls, ethyls,        propyls, isopropyls, —NH₂ amino groups, —NH₃ ⁺ ammonium groups,        or —SO₄ ²⁻ sulphate groups, and are in particular hydrogen atoms        or methyl groups,        said CD being alpha-cyclodextrin (α-CD) in the form of monomer,        at a concentration in the range from 1 to 150 g/L of aqueous        medium, in particular in the range from 1 to 50 g/L of aqueous        medium, and in particular equal to approximately 10 g/L of        aqueous medium,        said chitosans of formula I or II being    -   in aqueous solution at a concentration in the range from 1 to        150 g/L, in particular being in the range from 1 to 10 g/L, and        in particular being equal to approximately 1 or equal to        approximately 10 g/L,        the aqueous solvent being selected from pure water, an aqueous        solution with pH in the range from 1 to 7 or from 7 to 12, in        particular in the range from 5 to 7, or a physiological serum        solution optionally enriched with glucose or containing any        other excipient for pharmaceutical, cosmetic, agri-food or        veterinary use,    -   or in dispersion in an aqueous medium selected from pure water,        an aqueous solution with pH in the range from 1 to 7 or from 7        to 12, in particular in the range from 5 to 7, or a        physiological serum solution optionally enriched with glucose or        containing any other excipient for pharmaceutical, cosmetic,        agri-food or veterinary use, the weight of said polysaccharide        being in the range from 1 to 150 g/L of aqueous medium, in        particular being in the range from 1 to 10 g/L of aqueous        medium, and in particular being equal to 1 g/L or 10 g/L of        aqueous medium,    -   the percentage by weight of said polysaccharide being in the        range from 0.1% to 15%, and in particular from 0.5% to 5%,    -   the percentage by weight of the cyclodextrin being in the range        from 0.1% to 15%, and in particular from 0.5% to 5%,    -   the percentage by weight of water or of aqueous medium being in        the range from 70% to 99.9%, and in particular from 90% to 99%,        said mixing being carried out with stirring, in particular        magnetic, at a stirring speed in the range from 50 to 1000 rpm,        in particular at a stirring speed in the range from 100 to 500        rpm, and in particular at a stirring speed equal to        approximately 200 rpm,        at a temperature in the range from 4 to 60° C., in particular at        a temperature in the range from 10 to 35° C., and in particular        at a temperature equal to approximately 20° C.,        the duration of stirring being in the range from 24 hours to 15        days, in particular in the range from 24 hours to 48 hours, and        in particular equal to approximately 36 hours,        to obtain an inclusion complex in which the chitosan of formula        I or II and the cyclodextrin are bound non-covalently.

According to a particular embodiment, the method for preparing aninclusion complex comprises a step of mixing between

-   -   a chitosan of formula:

or a chitosan of formula:

in which

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

in which

-   -   R⁴ represents a hydrophobic group and is selected from:        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and bearing at least one C═C double bond, in            particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,            provided that R represents at least one group of formula

-   -   m represents the number of D-glucosamine units,    -   n represents the number of N-acetyl-D-glucosamine units,        provided that the percentage of m relative to the total number        of units is greater than 50%,    -   and a cyclodextrin (CD) of formula:

in which

-   -   p is an integer equal to 6,    -   R¹, R² and R³, which may be identical or different, in        particular identical, are hydrogen atoms, alkyl groups        comprising 1 to 3 carbon atoms, selected from methyls, ethyls,        propyls, isopropyls, —NH₂ amino groups, —NH₃ ⁺ ammonium groups,        or —SO₄ ²⁻ sulphate groups, and are in particular hydrogen atoms        or methyl groups,        said CD being alpha-cyclodextrin (α-CD) in the form of monomer,        at a concentration in the range from 0.01 to 9000 g/L of aqueous        medium, in particular in the range from 1 to 300 g/L of aqueous        medium, and in particular equal to approximately 200 g/L of        aqueous medium,        said chitosans of formula I or II being    -   in aqueous solution at a concentration in the range from 0.01 to        9000 g/L, in particular being in the range from 1 to 300 g/L,        and in particular being equal to approximately 200 g/L,        the aqueous solvent being selected from pure water, an aqueous        solution with pH in the range from 1 to 7 or from 7 to 14, in        particular in the range from 5 to 7, or a physiological serum        solution optionally enriched with glucose or containing any        other excipient for pharmaceutical, medical, paramedical,        medical-device, animal-feed, cosmetic, veterinary, agri-food,        pesticide, cosmetotextile, perfumery, and environmental use        (water purification for example) or in the paint, packaging,        building and/or car industry,    -   or in dispersion in an aqueous medium selected from pure water,        an aqueous solution with pH in the range from 0 to 7 or from 7        to 14, in particular in the range from 5 to 7, or a        physiological serum solution optionally enriched with glucose or        containing any other excipient for pharmaceutical, medical,        paramedical, medical-device, animal-feed, cosmetic, veterinary,        agri-food, pesticide, cosmetotextile, perfumery, and        environmental use (water purification for example) or in the        paint, packaging, building and/or car industry,        the weight of said polysaccharide being in the range from 0.01        to 9000 g/L of aqueous medium, in particular being in the range        from 1 to 60 g/L of aqueous medium, and in particular being        equal to 1 g/L or 10 g/L of aqueous medium,    -   the percentage by weight of said polysaccharide being in the        range from 0.01% to 90%, and in particular from 0.5% to 5%,    -   the percentage by weight of the cyclodextrin being in the range        from 0.01% to 90%, and in particular from 0.5% to 5%,    -   the percentage by weight of water or of aqueous medium being in        the range from 10% to 99.99%, and in particular from 50% to        99.5%,        said mixing being carried out with stirring, in particular        magnetic, at a stirring speed in the range from 10 to 1000 rpm,        in particular at a stirring speed in the range from 100 to 500        rpm, and in particular at a stirring speed equal to        approximately 200 rpm,        at a temperature in the range from 1 to 100° C., in particular        at a temperature in the range from 10 to 35° C., and in        particular at a temperature equal to approximately 20° C.,        the duration of stirring being in the range from 12 hours to 15        days, in particular in the range from 24 hours to 96 hours, and        in particular equal to approximately 72 hours,        to obtain an inclusion complex in which the chitosan of formula        I or II and the cyclodextrin are bound non-covalently.

In another aspect, the invention also relates to the particles obtainedby the methods described above.

The invention also relates to a chitosan bearing hydrophobic groupshaving as formula II:

in which

-   -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 20            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 20 carbon            atoms and bearing from 1 to 4 C═C double bonds, conjugated            or not, in particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,    -   m represents the number of deacetylated units,    -   n represents the number of acetylated units,        provided that the degree of deacetylation representing the        percentage of m relative to the total number of units is greater        than 50%.

The invention also relates to a chitosan bearing hydrophobic groupshaving formula II:

in which

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

in which

-   -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and bearing at least one C═C double bond, in            particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,            provided that R represents at least one group of formula

-   -   m represents the number of D-glucosamine units,    -   n represents the number of N-acetyl-D-glucosamine units,        provided that the degree of deacetylation (DDA) representing the        percentage of m relative to the total number of units is greater        than 50%.

The invention also relates to a carrageenan bearing hydrophobic groupshaving formula I and/or II:

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

in which

-   -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and bearing at least one C═C double bond, in            particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,            provided that R represents at least one group of formula

The invention also relates to a heparin bearing hydrophobic groupshaving the formula:

in which

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

R⁴ represents

-   -   a linear or branched alkyl group containing from 1 to 1000        carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃        groups,    -   a linear or branched alkenyl group containing 2 to 1000 carbon        atoms and bearing at least one C═C double bond, in particular        the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or —(CH₂)₇—CH═CH—(CH₂)₇—CH₃        groups,        provided that R represents at least one group of formula

The invention also relates to an amylopectin bearing hydrophobic groupshaving the formula:

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

in which

-   -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and bearing at least one C═C double bond, in            particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,            provided that R represents at least one group of formula

The invention also relates to a pullulan bearing hydrophobic groupshaving the formula:

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

in which

-   -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and bearing at least one C═C double bond, in            particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,            provided that R represents at least one group of formula

The invention also relates to a dextran bearing hydrophobic groupshaving the formula:

in which

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

in which

-   -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 20            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 20 carbon            atoms and bearing from 1 to 4 C═C double bonds, conjugated            or not, in particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,            provided that R represents at least one group of formula

The invention also relates to a pectin bearing hydrophobic groups havingthe formula:

in which

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

-   -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 20            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 20 carbon            atoms and bearing from 1 to 4 C═C double bonds, conjugated            or not, the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups.            provided that R represents at least one group of formula

The invention also relates to hyaluronic acid bearing hydrophobic groupshaving the formula:

in which

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

-   -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and bearing at least one C═C double bond, in            particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,            provided that R represents at least one group of formula

The invention also relates to a chitin bearing hydrophobic groups havingthe formula:

-   -   R represents        -   a hydrogen atom, or        -   a group of formula

in which

-   -   R⁴ represents        -   a linear or branched alkyl group containing from 1 to 1000            carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃            groups,        -   a linear or branched alkenyl group containing 2 to 1000            carbon atoms and bearing at least one C═C double bond, in            particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or            —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,            provided that R represents at least one group of formula

-   -   m represents the number of N-glucosamine units,    -   n represents the number of N-acetyl-glucosamine units,        provided that the percentage of units n relative to the total        number of units is greater than 50%.

In a preferred embodiment, the linear or branched alkyl group containingfrom 1 to 20 carbon atoms and the linear or branched alkenyl groupcontaining 2 to 20 carbon atoms and bearing from 1 to 4 C═C doublebonds, conjugated or not, are selected from the fatty acids.

The invention also relates to a method for preparing an encapsulationsystem comprising a step of mixing particles containing inclusioncomplexes according to the invention,

with a substance used for its properties in the pharmaceutical, medical,paramedical, medical-device, animal-feed, cosmetic, veterinary,agri-food, pesticide, cosmetotextile, perfumery, and environmentalfields (water purification for example) or in the paint, packaging,building and/or car industry,said substance being dissolved beforehand in an aqueous medium such aswater, physiological serum, said medium optionally being enriched withglucose or containing any other excipient for pharmaceutical, medical,paramedical, medical-device, animal-feed, cosmetic, veterinary,agri-food, pesticide, cosmetotextile, perfumery, and environmental use(water purification for example) or in the paint, packaging, buildingand/or car industry, said medium optionally containing a co-solventselected from ethanol or acetone, or a surfactant selected from thepolysorbate derivatives, in particular Tween 80 or Tween 40, to obtainparticles containing inclusion complexes containing said substance.

According to a particular embodiment, the method for preparing anencapsulation system according to the invention comprises a step ofmixing:

-   -   a polysaccharide comprising hydrophobic groups fixed covalently        to the polysaccharide by a nitrogen atom and by oxygen atoms of        said polysaccharide,        said polysaccharide being selected from chitosan, dextran,        hyaluronic acid, amylose, amylopectin, pullulan, heparin,        chitin, cellulose derivatives, heparan sulphate, dermatan        sulphate, keratan sulphate, chondroitin sulphate, cellulose        sulphate, dextran sulphate, dextrin sulphate, starch, pectin,        the alginates, the carrageenans, fucan, curdlan, xylan,        polyguluronic acid, xanthan, arabinan, polymannuronic acid, and        derivatives thereof, and is in particular chitosan,    -   a cyclodextrin designated above,    -   a substance used for its properties in the pharmaceutical,        medical, paramedical, medical-device, animal-feed, cosmetic,        veterinary, agri-food, pesticide, cosmetotextile, perfumery, and        environmental fields (water purification for example) or in the        paint, packaging, building and/or car industry,    -   the percentage by weight of said solvent being in the range from        0 to 50% and in particular equal to approximately 25%,    -   the percentage by weight of said polysaccharide being in the        range from 0.1% to 15%, and in particular from 0.5% to 5%,    -   the percentage by weight of the cyclodextrin being in the range        from 0.1% to 15%, and in particular from 0.5% to 5%,    -   the percentage by weight of water or of aqueous medium being in        the range from 30% to 99.9%, and in particular from 90% to 99%,        to obtain an inclusion complex containing said substance.

The mixing step in the method for preparing an encapsulation systemcomprises a step of dissolving the active ingredient in an aqueousmedium, followed by addition of the amphiphilic polysaccharide andα-cyclodextrin to the medium. The mixture is stirred magnetically for 3days. In practice, however, it is possible to mix all the components atthe same time.

The particles may then be isolated

-   -   either by sedimentation, or by centrifugation if they are        microparticles (hydrodynamic diameter greater than a        micrometre),    -   or by ultracentrifugation or by membrane separation methods such        as ultrafiltration and microfiltration, if they are        nanoparticles (hydrodynamic diameter less than a micrometre).

DESCRIPTION OF THE FIGURES

FIG. 1 shows the IR spectrum characteristic of chitosan N-acylated witholeic acid (1) in comparison with that of native chitosan (2).

FIG. 2 shows the formula of unmodified chitosan and its ¹H-NMR spectrum(300 MHz) in DC1 at 1% v/v in D₂O. The hydrogen atoms are numbered onthe formula of the compound, these numbers being shown on the peaks ofthe NMR spectrum.

FIG. 3 shows the ¹H-NMR spectrum (300 MHz) of chitosan after N-acylationwith oleic acid dissolved in DC1 at 1% v/v in D₂O.

FIG. 4 shows the IR spectrum of O-palmitoyl-chitosan (1) in comparisonwith native chitosan (2).

FIG. 5 shows the proton NMR spectrum in DMSO-d₆, ofO-palmitoyl-chitosan.

FIG. 6 shows the IR spectrum of O-palmitoyl-pullulan (1) in comparisonwith that of native pullulan (2).

FIG. 7 shows the IR spectrum of O-palmitoyl-amylopectin (1) incomparison with that of native amylopectin (2).

FIG. 8 shows the IR spectrum of O-palmitoyl-dextran (1) in comparisonwith that of native dextran (2).

FIG. 8 a shows the IR spectrum of O-palmitoyl-dextran.

FIG. 9 shows the IR spectrum of chitin esterified with palmitic acid (1)in comparison with that of native chitin (2).

FIG. 9 a shows the IR spectrum of O-palmitoyl-chitin.

FIG. 10 shows the ¹³C-NMR spectrum in the solid state ofO-oleoyl-chitin.

FIG. 11 shows the IR spectrum characteristic of O-palmitoyl-heparin (1)in comparison with native heparin (2).

FIG. 12 shows the IR spectrum characteristic of O-palmitoyl-carrageenan(1) in comparison with native carrageenan (2).

FIG. 13 shows the IR spectrum characteristic of O-palmitoyl-hyaluronicacid (1) in comparison with native hyaluronic acid (2).

FIG. 14 shows the images of different preparations of particles,obtained by transmission electron microscopy.

FIG. 15 shows the α, β and γcyclodextrins. In the figure, the dimensionsare given in Angströms.

FIG. 16 shows the effect of the concentration of O-palmitoyl-heparin DS1% on the D_(h) of the nanoparticles.

FIG. 17 shows the effect of the concentration of α-CD on the D_(h) ofthe microparticles consisting of O-palmitoyl-heparin DS2.

FIG. 18 shows TEM observation of the particles consisting ofO-palmitoyl-heparin DS2.

FIG. 19 shows the effect of the concentration of α-CD on the D_(h) ofthe microparticles consisting of O-palmitoyl-carrageenan DS1.

FIG. 20 shows the effect of the concentration of O-palmitoyl-carrageenanDS1 on the D_(h) of the microparticles.

FIG. 21 shows the effect of the concentration of α-CD on the D_(h) ofthe particles of O-palmitoyl-carrageenan DS2.

FIG. 22 shows the effect of the concentration of O-palmitoyl-carrageenanDS3 on the D_(h) of the microparticles formed.

FIG. 23 (1) shows an image of the preparation α-cyclodextrin/nativechitosan/water (10/1/89)% indicating absence of formation ofnanoparticles or microparticles, obtained by transmission electronmicroscopy.

FIG. 23 (2) shows nanoparticles consisting of α-cyclodextrin/MC6/water(10/1/89)%.

FIG. 24 (1) shows an image of the nanoparticles at the laboratory scale,obtained by transmission electron microscopy.

FIG. 24 (2) shows an image of the nanoparticles at the pilot scale,obtained in transmission electron microscopy.

EXAMPLES Meanings of the Abbreviations Used:

-   CD: cyclodextrin-   CDCl₃: deuterated chloroform-   MC: modified chitosan-   D₂O: deuterated water-   Da: unit of molecular weight in daltons, which corresponds to g/L-   DCl: deuterated hydrochloric acid-   DDA: degree of deacetylation-   P_(h): hydrodynamic diameter-   DMF: dimethylformamide-   DMSO-d6: deuterated dimethylsulphoxide-   DS: degree of substitution-   EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-   HSV: human herpes simplex virus-   HPV: human papillomavirus-   IR: infrared-   kDa: kilodalton-   kHz: kilohertz-   TEM: transmission electron microscopy-   MW: molecular weight-   MHz: megahertz-   NaNO₂: sodium nitrite-   Q.S.: sufficient quantity-   NMR: nuclear magnetic resonance-   rpm: revolutions per minute-   RSV: respiratory syncytial virus-   δ: proton shift, expressed in ppm-   MW: molecular weight-   PA: palmitic acid-   OA: oleic acid

Characteristics/Types of Analytical Equipment Used:

¹H-NMR (Bruker ARX, 300 MHz or 500 MHz):

All the N- or O-acylated chitosan compounds obtained were analysed byproton NMR. Depending on the solubility of the acylated polysaccharides,various solvents may be used, such as deuterated water (D₂O) with 1% v/vof DC1, deuterated chloroform (CDCl₃), deuterated dimethylsulphoxide(DMSO-d6). The spectra are recorded at ambient temperature. If theacylated polysaccharide is sparingly soluble or if the viscosity ishigh, the samples are heated to 85° C. and an electromagnetic field of500 MHz is used.

Characterization of the solid by ¹³C-NMR:

The spectra were recorded on an AVANCE II BRUKER spectrometer, usingspecialized techniques for NMR of solids, in particular magic anglespinning (MAS) and transfer of polarization from ¹H to ¹³C (CP) to avoidthe drawbacks connected with the sometimes very long relaxation times inthe solid state. The work was carried out at ωL=500 MHz (¹H) and 125.77MHz (¹³C); the ¹H/¹³C contact time was fixed at 1.5 ms. These sampleswere rotated at the magic angle at a frequency of 10 kHz, using a 4-mmrotor.

IR Spectra (ATR-FTIR, FT/IR-4100 Spectrometer, JASCO):

the principle consists of bringing a crystal (diamond) into contact withthe sample to be analysed, before the infrared beam is passed through.

Measurements of Particle Size:

The particle sizes were evaluated from the hydrodynamic diameter. Theaverage hydrodynamic diameters of the nanoparticles and microparticleswere measured with a Zetasizer nanoseries Nano-ZS90 from the companyMalvern instruments SA (Orsay, France) by quasi-elastic lightscattering. The samples were diluted beforehand, taking 30 μL ofsuspension of nanoparticles or microparticles and diluting it in 1 mL ofMilliQ® water. The measurements of the hydrodynamic diameters of themicroparticles were repeated with a laser granulometer (MasterSizer2000) from the company Malvern instruments SA (Orsay, France).

Transmission Electron Microscopy

(TEM) was used for observing the microparticles and nanoparticles usinga Jeol 1400 60 kV microscope and a camera.

Lyophilization of the Polysaccharides Grafted with Hydrophobic Groups:

Certain derivatives of synthesized polysaccharides were lyophilizedusing an Alpha 1-2 lyophilizer (Avantec, France) for 48 hours after thesolutions had been frozen for at least 12 hours.

Details of the Products Used:

The active ingredients, vitamin B6, paracetamol, ketoprofen,indometacin, caffeine were obtained from the company INRESA, Bartenheim,France. Parsol 1789 is from the company DSM Nutritional Products,Germany.

All the solvents: anhydrous dimethylformamide, dichloromethane, diethylether, ethanol, methanol, ammonia, glacial acetic acid (98% (w/w)) wereobtained from VWR, France. Chitosan (of medium viscosity, with molecularweight MW=250 kDa), pullulan, amylopectin, dextran, i-carrageenan,heparin, hyaluronic acid, N-(3-dimethylaminopropyl)-N-ethylcarbodiimidechloride (EDCI), palmitic acid (99% (w/w)), oleic acid (>98% (w/w)),sodium bicarbonate, sodium hydroxide, sodium chloride, sodium nitrite(NaNO₂), methanesulphonic acid, palmitoyl chloride, oleoyl chloride,deuterated water, deuterated hydrochloric acid (DC1), deuteratedchloroform (CDCl₃), deuterated dimethylsulphoxide (DMSO-d₆), anhydrouspyridine, triethylamine, Sudan III, hydroxypropyl-β-cyclodextrin(HP-β-CD) were supplied by Sigma-Aldrich Chemical Co, Saint QuentinFallavier, France. The castor oil was obtained from the company Croda,France. α-Cyclodextrin (α-CD), methyl-β-cyclodextrin (Me-β-CD) Rameb®,degree of substitution ˜1.8, MW=1320 g/mol, was purchased from Cyclolab(Budapest, Hungary).

The other chitosans (20 and 145 kDa) were obtained afterdepolymerization of the commercial chitosan MW=250 kDa according to thetechnique developed by Huang et al. (Pharmaceutical Research, 2004,21(2), 344): 2 g of commercial chitosan is solubilized overnight in 100mL of acetic acid (6% v/v), this chitosan solution is then depolymerizedby chemical reaction for 1 h with 10 mL of NaNO₂ (85 mg/mL). Thedepolymerized chitosan is then precipitated by increasing the pH to 9using sodium hydroxide solution (4 mol/L) and recovered by filtration(sintered glass filter No. 4, under vacuum). It is then dissolved in 20mL of acetic acid (0.1 mol/L) and dialysed (dialysis membraneSpectra/Por, MWCO 3500, batch 3228543, Spectrum laboratories Inc®)against 1 L of distilled water twice for 1.5 h and then overnight, andthen lyophilized.

The molecular weight of the depolymerized chitosan is determined bycapillary viscosimetry. The flow time (t), in a μ-Ubbelohde microtube(type 53710/I, No. 1016187, R=0.01022 mm²/s², Schott Geräte), ofsolutions of chitosan in a mixture of acetic acid 0.1 mol/L and NaCl 0.2mol/L at different concentrations (c=0.25/0.50/1.00/1.50/2.00 g/L) ismeasured at 20° C. (bath CT1450 Schott Gerate and cooling system CK100Schott Gerate) using an AVS400 viscosimeter (Schott Geräte). For eachconcentration, the equilibration time is 5 min and five successivemeasurements are performed. Based on the results obtained, the intrinsicviscosity [η] is deduced, taking the ordinate at the origin of thestraight line

$\frac{t - t_{0}}{t_{0}C} = {f(C)}$

with t₀ the flow time of the mixture of acetic acid 0.1 mol/L and NaCl0.2 mol/L. The molecular weight is then calculated using theMark-Houwink-Sakurada equation η=KM_(w) ^(a), with K=1.81×10⁻⁶ anda=0.93.

Synthesis of the Polysaccharides Bearing Hydrophobic Groups Example 1Synthesis and Characterization of N-Acylated Chitosan

The protocol consists of dissolving 1 g of chitosan (MW=20, 145, 250kDa), the degree of deacetylation (DDA) of which is equal to 85%, in anaqueous solution of acetic acid at 1% v/v (100 mL) diluted with methanol(75 mL). Oleic acid or palmitic acid is dissolved in 10 mL of methanoland is added to the chitosan solution. A number of moles of EDCI equalto that of the fatty acid is added dropwise to the mixture of fatty acidand chitosan, under continuous magnetic stirring. After reaction for 24hours, the product is precipitated from a methanol/ammonia mixture, 7/3v/v. The precipitate is filtered on a sintered glass filter and washedsuccessively with water, methanol and then diethyl ether. Finally, theproduct is dried under vacuum for 48 hours.

During this reaction, several parameters were varied. In this way it ispossible to obtain several types of N-acylated chitosans depending onthe type of grafted fatty acid, the degree of grafting (degree ofsubstitution) and the molecular weight of the chitosan. The IR spectraof the grafted chitosans obtained were recorded. The two curves in FIG.1 reveal a very wide band between 3430 cm⁻¹ and 3440 cm⁻¹ correspondingto the OH groups (FIG. 1). The native chitosan has weak absorption atthe level of the vibration of the NH groups around 1578 cm⁻¹. AfterN-acylation, it is to be noted that there is weak absorption of itsderivatives at bands 3000 cm⁻¹ and 3600 cm′ and the appearance of twobands 1636 cm⁻¹ and 1657 cm⁻¹, characteristic of the carbonyl groups andsecondary amides respectively and providing good confirmation of theformation of the amide bond. As for the bands at 2922 cm⁻¹, 2853 cm⁻¹,1457 cm⁻¹ and 1198 cm⁻¹, they correspond to the alkyl chains of thefatty acid.

Comparison of the proton NMR spectra (FIGS. 2 and 3) shows an additionalsignal with respect to the spectrum of the unmodified chitosan at6=1.017 ppm. It corresponds to the signal of the methyl group at the endof the fatty acid chain.

Example 2 Characteristics of the Modified Chitosans MC1-MC9

The characteristics of the modified chitosans MC1-MC9 are presented inTable 1 below. The degree of substitution was calculated from theresults of elemental analysis of the N-acylated chitosan and of nativechitosan.

TABLE 1 Characteristics of the N-acylated chitosans Grafted MW of Degreeof Modified hydrophobic chitosan used substitution chitosan group (kDa)(%) MC1 Oleic acid 250 1.19 MC2 Oleic acid 250 1.67 MC3 Oleic acid 2507.43 MC4 Oleic acid 145 13.47 MC5 Oleic acid 20 5.61 MC6 Oleic acid 206.35 MC7 Palmitic acid 250 0.55 MC8 Palmitic acid 250 13.12 MC9 Palmiticacid 250 17.01

Example 3 Preparation of the Samples of Modified Chitosans, MC1-MC9.Characterization by Proton NMR

For characterization of the N-acylated chitosan by proton NMR, asolution of polymer at a concentration equal to 5 g/L is prepared indeuterated water (D₂O) in the presence of deuterated hydrochloric acid(DC1). This step allows exchange of the labile protons of the hydroxylgroups with deuterium atoms. Since the labile protons of the hydroxylgroups all resonate at the same frequency, exchanging them withdeuterium atoms makes it possible to remove the residual signal of lightwater. In order to lower the viscosity, the experiments were recorded ata temperature of 85° C. with an acquisition number and a relaxationdelay of 5 and 1 seconds respectively.

¹H-NMR (300 MHz) unmodified chitosan: the correspondence of each protonshift is indicated in FIG. 2.

¹H-NMR (300 MHz) modified chitosan: an additional peak relative to thespectrum of unmodified chitosan appears for a chemical shift equal toδ₈=1.017 ppm (FIG. 3). It corresponds to the signal of the fatty acidchain.

Example 4 Synthesis and Characterization of O-Acylated Chitosan

Chitosan (250 kDa, 2 g) is dissolved in 20 mL of methanesulphonic acid,at ambient temperature under continuous magnetic stirring for one hour.The acid chloride (oleic or palmitic) is then added to the reactionmixture. After 5 hours, the mixture is cooled down in an ice bath tostop the reaction; a precipitate forms. The precipitate is dialysed for12 hours, and then neutralized with sodium bicarbonate. It is thendialysed for 48 hours and lyophilized.

The IR spectrum (FIG. 4) of O-palmitoyl-chitosan shows a bandcharacteristic of the carbonyls of an ester group at 1700 cm⁻¹, andbands at 2916 cm⁻¹, 2849 cm⁻¹ corresponding to the alkyl chains of thefatty acid. The proton NMR spectrum (FIG. 5) compared with the spectrumof native chitosan (FIG. 2), shows the appearance of new peaks includingthose characteristic of the CH₃ group at the end of the chain at 0.88ppm and of the proton of the methylene groups CH₂ close to the COfunction at 2.8 ppm.

Example 5 Table 2, Characteristics of the Modified Chitosans MC10-MC12

The characteristics of the modified chitosans MC10-MC12 are presented inTable 2 below. The degree of substitution was calculated from theresults of elemental analysis of the O-acylated chitosan and of nativechitosan.

TABLE 2 Characteristics of the O-acylated chitosans Grafted MW of Degreeof Modified hydrophobic chitosan used substitution chitosan group (kDa)(%) MC10 Oleic acid 250 1.41 MC11 Palmitic acid 250 2.25 MC12 Palmiticacid 250 4.64

Example 6 Synthesis and Characterization of Pullulan Grafted withPalmitic Acid

O-Palmitoyl-pullulan was prepared according to the reference of Sunamotoet al. (Sunamoto J., Sato T., Taguchi T., Hamazaki H., Naturallyoccurring polysaccharide derivatives which behave as an artificial cellwall on an artificial liposome, Macromolecules, 1992, 25, 5665-5670).Pullulan (5 g) is dissolved in anhydrous dimethylformamide (55 mL) at60° C. 5 mL of anhydrous pyridine and palmitoyl chloride (5 equivalentsper unit of glucose triholoside) are added to the solution obtained. Thereaction mixture is stirred at 60° C. for 2 h and then for 1 h atambient temperature. The mixture is poured into ethanol (350 mL). Theprecipitate obtained is extracted and washed with ethanol and then withdiethyl ether. The white solid obtained is dried under vacuum.

IR spectrum of O-palmitoyl-pullulan: band at 1739 cm⁻¹ corresponding tothe carbonyl of the ester group (FIG. 6) and bands at 2916 cm⁻¹, 2849cm⁻¹, 1467 cm⁻¹ and 1197 cm⁻¹, corresponding to the alkyl chains of thefatty acid.

Example 7 Synthesis and Characterization of Derivatives of AmylopectinGrafted with Palmitic Acid

Amylopectin (5 g) is mixed with anhydrous dimethylformamide. The mixtureis stirred at 70° C. until the polysaccharide has completely dissolved.Then anhydrous triethylamine and palmitoyl chloride are added and thenheated under stirring for 2 h. The solution is then diluted in methanol,resulting in precipitation of O-palmitoyl-amylopectin. The white solidobtained is filtered and then dried under vacuum.

IR spectrum of O-palmitoyl-amylopectin: band at 1739 cm⁻¹ correspondingto the carbonyl of the ester group (FIG. 7) and in particular bands at2916 cm⁻¹, 2849 cm⁻¹, 1462 cm⁻¹ corresponding to the alkyl chains of thefatty acid.

Example 7a Synthesis and Characterization of Derivatives of AmylopectinGrafted with Palmitic Acid

O-Palmitoyl-amylopectin was prepared according to the reference ofSunamoto et al. (Sunamoto J., Sato T., Taguchi T., Hamazaki H.,Naturally occurring polysaccharide derivatives which behave as anartificial cell wall on an artificial liposome, Macromolecules, 1992,25, 5665-5670). Amylopectin (5 g) is mixed with 55 mL of anhydrousdimethylformamide at 60° C., under continuous magnetic stirring. 5 mL ofanhydrous pyridine, and 1.2 mL of anhydrous DMF containing palmitoylchloride are added to the solution obtained. The quantity of palmitoylchloride was varied according to the desired degree of substitution. Thereaction mixture is stirred at 60° C. for 2 h and then for 1 h atambient temperature. The mixture is poured into 350 mL of ethanol. Thewhite solid obtained is dried under vacuum.

Example 8 Synthesis and Characterization of Derivatives of DextranGrafted with Palmitic Acid

Dextran is suspended in anhydrous dimethylformamide. The mixture isstirred at 70° C. until the polysaccharide has completely dissolved.Then anhydrous triethylamine and palmitoyl chloride are added and thenheated under stirring for 2 h. The solution is then diluted in methanol,resulting in the precipitation of O-palmitoyl-dextran. The white solidobtained is filtered and then dried under vacuum.

IR spectrum of O-palmitoyl-dextran: band at 1739 cm⁻¹ corresponding tothe carbonyl of the ester group (FIG. 8) and in particular bands at 2914cm⁻¹, 2853 cm⁻¹ corresponding to the alkyl chains of the fatty acid.

Example 8a Synthesis and Characterization of Derivatives of DextranGrafted with Palmitic Acid

O-Palmitoyl-dextran was prepared according to the protocol described bySunamoto et al. (Sunamoto J., Sato T., Taguchi T., Hamazaki H.,Naturally occurring polysaccharide derivatives which behave as anartificial cell wall on an artificial liposome, Macromolecules, 1992,25, 5665-5670). Dextran (5 g) is mixed with 55 mL of anhydrousdimethylformamide at 60° C. 5 mL of anhydrous pyridine and palmitoylchloride are added to the solution obtained. The reaction mixture isstirred at 60° C. for 2 hours and then for 1 hour at ambienttemperature. The mixture is poured into 350 mL of ethanol. Theprecipitate obtained is extracted and washed with ethanol and then withdiethyl ether. The white solid obtained is dried under vacuum.

IR spectrum of O-palmitoyl-dextran (FIG. 8 a): bands at 2914 cm⁻¹ and at2848 cm′ corresponding to the alkyl chains of the fatty acid.

Example 9 Synthesis and Characterization of O-Oleoyl-Chitin andO-Palmitoyl-Chitin

Synthesis: Chitin was esterified with oleic acid by the method describedin the reference (Yang B. Y., Ding Q., Montgomery R., Preparation andphysical properties of chitin fatty acids esters, Carbohydrate Research,2009, 344 (3), 336-342). Chitin (7.5 g), dried beforehand under vacuumat 60° C. for 16 hours, is introduced into a mixture of trifluoroaceticacid (75 mL) and palmitic acid or oleic acid (28 g). The mixture is thenheated at 70° C. under continuous stirring for 30 min, and then cooleddown to ambient temperature. Next, 300 mL of cold absolute ethanol (−20°C.) is added, and the mixture is concentrated in a rotary evaporator ata temperature of 20° C. under vacuum. The product obtained is dispersedin absolute ethanol and heated to 80° C. After decanting and removingthe supernatant, the end product is dried and dissolved in 30 mL of DMFand then centrifuged at 3600 g.

Esterification of chitin with palmitic acid or oleic acid is carried outin the presence of trifluoroacetic acid, which is known to be a promoterof esterification for other polysaccharides, in particular starch citedin the reference (Yang B. Y., Montgomery R., Acylation of Starch usingTrifluoroacetic Anhydride Promoter, Starch-Stärke, 2006, 58 (10),520-526). In fact, trifluoroacetic acid leads to the formation of theacid anhydride, which is much more reactive than the carboxylic aciditself. Being strongly electronegative and highly acidic,trifluoroacetic acid leads to the protonation of the amine functions,thus permitting preferential esterification on the alcohol functions.

Characterization of O-oleoyl-chitin and O-palmitoyl-chitin:

Analysis by infrared spectroscopy is a quick and efficient method foridentifying the chemical groups of the esterified chitin in comparisonwith the spectrum of native chitin. The spectrum shown in FIG. 9 istypical of chitin; the frequency of the carbonyl regions (CO) of theamides between 1600 cm⁻¹ and 1500 cm⁻¹ is of high intensity. The bandcorresponding to amide I is divided into two peaks at 1654 cm⁻¹ and 1619cm⁻¹. The band corresponding to amide II is single and is at 1556 cm⁻¹.

Regarding the spectrum obtained for O-palmitoyl-chitin (FIG. 9 a),greater absorption is observed for the bands at 1649 cm⁻¹ and 1554 cm⁻¹,which explains the presence of the carbonyl groups CO and amide II, thusdemonstrating N-acylation of the free amine functions of the chitin.FIG. 9 a (1) also shows bands at 2916 cm⁻¹ and at 2849 cm⁻¹corresponding to the alkyl chains of the fatty acid.

Characterization by ¹³C-NMR of the solid of the chitin esterified witholeic acid is particularly suitable for characterization of thisderivative, which is insoluble in water and in the majority of organicsolvents. The spectrum presented in FIG. 10 shows the presence of 9resonance peaks with the values presented in Table 3 below.

TABLE 3 Shifts obtained in NMR of the solid carbon of O-oleoyl- chitinexpressed in ppm and the corresponding functions Function Shift (ppm)═CH—, —CH₂—, —CH₃ 14.29, 22.93, 29.81 C2 55.35 C6 60.96 C3-C5 73.87 C483.85 C1 104.03 C═O amide and ester 173.79

Example 10 Synthesis of Heparin Grafted with Palmitic Acid DS1

Introduce 1 g of heparin into 11 mL of anhydrous DMF. Heat gradually at60° C. under magnetic stirring. Add 5 mL of anhydrous pyridine and then2.5 g of palmitoyl chloride in 6 mL of anhydrous DMF. Stir the mixtureunder magnetic stirring for 2 h at 60° C. and then for 1 h at ambienttemperature. Pour the mixture into 100 mL of cold ethanol in order toobtain a precipitate. Extract and wash the precipitate with 100 mL ofethanol and then 100 mL of diethyl ether.

Example 10a Synthesis and Characterization of Heparin Grafted withPalmitic Acid DS2

Synthesis: Heparin (2 g) is dissolved in 10 mL of dichloromethane and 2mL of palmitic acid chloride contained in a flask. The flask is placedunder reflux under continuous magnetic stirring for 72 h at ambienttemperature. Next, 20 mL of a solution of sodium acetate in methanol isadded to the reaction mixture. The precipitate formed is recovered on asintered glass filter of porosity No. 4, then washed with 100 mL ofmethanol and then 100 mL of acetone. The solid is dried under vacuum atambient temperature. The heparin esterified with palmitic acid ispurified by dissolving in 10 mL of distilled water and the addition ofNaCl until a concentration of NaCl of 10% w/v is reached. Next, 20 mL ofmethanol is added and the precipitate formed is recovered on a sinteredglass filter and then washed with 100 mL of ethanol and then 100 mL ofacetone. The solid is dried under vacuum at ambient temperature.

Characterization of O-palmitoyl-heparin: Analysis by infraredspectroscopy (FIG. 11) showed the presence of bands at 1620 and 1724cm⁻¹, which correspond to the carbonyl groups of the ester function. Thebands at 1135 and 1187 cm⁻¹ correspond to the C—O groups of the esterfunction. The spectrum of O-palmitoyl-heparin also showed bands at 2849and 2916 cm⁻¹ corresponding to the alkyl groups of palmitic acid.

Example 11 Synthesis and characterization of carrageenan grafted withpalmitic acid

Synthesis: Carrageenan (2 g) is suspended in 10 mL of dichloromethaneand 1, 2 or 3 mL of palmitic acid chloride contained in a flask. TheO-palmitoyl-carrageenans obtained are designated DS1, DS2 and DS3according to the quantity of acid chloride used in the reaction. Theflask is placed under reflux under continuous magnetic stirring for 72 hat 30° C. Next, the solid is recovered on a sintered glass filter andthen washed twice with 100 mL of ethanol. The solid is dried undervacuum at ambient temperature for 12 h. Infrared characterization (FIG.12): Characterization of O-palmitoyl-carrageenan by infraredspectroscopy showed the presence of bands at 1699 cm⁻¹ and 1739 cm⁻¹,which correspond to the carbonyl groups of the ester function. The bandsat 1047 cm⁻¹ and 1207 cm⁻¹ correspond to the C—O groups of the esterfunction. The spectrum of O-palmitoyl-carrageenan also showed bands at2848 cm⁻¹ and 2916 cm⁻¹ corresponding to the alkyl groups of palmiticacid.

Example 12 Synthesis and Characterization of Hyaluronic Acid Graftedwith Palmitic Acid

Hyaluronic acid (2 g) is mixed with 10 mL of dichloromethane and 2 mL ofpalmitic acid chloride contained in a flask. The flask is placed underreflux under continuous magnetic stirring for 5 days at 30° C. Next, thesolid is recovered on a sintered glass filter and then washed twice with100 mL of acetone. The solid is dried under vacuum at ambienttemperature for 12 h.

Infrared characterization (FIG. 13): Characterization ofO-palmitoyl-hyaluronic acid by infrared spectroscopy showed the presenceof bands corresponding to the carbonyl groups of the ester function. Thebands at 1048 and 1207 cm⁻¹ correspond to the C—O groups of the esterfunction. The spectrum of O-palmitoyl-hyaluronic acid also showed bandsat 2848 and 2915 cm⁻¹ corresponding to the alkyl groups of palmiticacid.

Example 13 Formation of Microparticles and Nanoparticles fromα-Cyclodextrin and Acylated Polysaccharides: N-Palmitoyl-Chitosan andO-Oleoyl-Chitosan

The microparticles and nanoparticles were formed from α-cyclodextrin andpolysaccharide grafted with fatty acids. The examples of thepolysaccharides used are presented in the table below. The protocolconsists of weighing the α-cyclodextrin and the O- or N-acylatedpolysaccharide in a small flask. Next, distilled water is added to themixture of α-cyclodextrin and the O- or N-acylated polysaccharide. Themixture is magnetically stirred for 3 days.

TABLE 4 Sizes of the particles obtained from N-acylated or O-acylatedchitosan Percentage Percentage Molecular by weight of by weightPercentage Particle weight Fatty DS amphiphilic of α- by weight sizepolysaccharide (kDa) acid (%) polysaccharide cyclodextrin of water (nm)*N-acylated 250 PA 13.12 1 10 89 7320 ± 1823 chitosan O-acylated 250 OA4.64 1 10 89 2090 ± 367  chitosan *Hydrodynamic diameter expressed byvolume.

An example of an image of particles observed using transmission electronmicroscopy is shown in FIG. 14.

Example 14 Encapsulation of Water-Soluble Active Ingredients

The protocol adopted for encapsulating the hydrophilic activeingredients is to dissolve the active ingredient in water at an initialconcentration as indicated in Table 5 and then adding the amphiphilicpolysaccharide and the α-cyclodextrin. The mixture is magneticallystirred for 3 days. After this mixing for 3 days, the concentration ofthe active ingredient that has not been encapsulated is determined inthe supernatant of the preparation. The supernatant is separated eitherby simple sedimentation or by centrifugation of the microparticles, orafter ultracentrifugation in the case of the nanoparticles.

TABLE 5 Example of active molecules encapsulated. Initial Encapsu-Polysac- concentration of Concentration lation charide active moleculesencapsulated yield used (g/L) (g/L) (%) Vitamin B6 MC8 194 104.65 53.94Vitamin B6 MC9 2.31 1.52 65.82 Paracetamol MC11 2.34 0.78 33.33 CaffeineMC8 21.7 3.15 17.53 Caffeine MC6 21.7 3.59 16.55 Caffeine MC9 2.33 0.5122.23

Example 15 Preparation and Characterization of Nanoparticles Composed of0-Palmitoyl-Heparin DS1

The protocol consists of weighing O-palmitoyl-heparin DS1 andα-cyclodextrin in a flask. Next, water is added to the mixture. Thewhole is mixed for 72 h at ambient temperature using a magnetic stirringbar. The concentrations of O-palmitoyl-heparin DS1 and of α-cyclodextrinare shown in Table 6. The results of the measurements of thehydrodynamic diameters are shown in Table 6 and FIG. 16.

TABLE 6 Effect of varying the concentration of O-palmitoyl-heparin onthe hydrodynamic diameter of the nanoparticles composed of heparin DS2Concen- Concen- Concen- tration of tration tration O-palmitoyl- ratioα-CD/O- of α-CD heparin palmitoyl- Name (g/L) DS1 (g/L) heparin DS1D_(h) (nm) Hep1 0 10 0 No formation of particles Hep2 100 10 10 344 ±105 Hep3 100 5 20 344 ± 128 Hep4 100 2.5 40 659 ± 260

Example 16 Preparation and Characterization of Nanoparticles Consistingof O-Palmitoyl-Heparin DS2

O-Palmitoyl-heparin DS2 and α-cyclodextrin were weighed in a flask.Next, water is added to the mixture. The whole is mixed for 72 h atambient temperature using a magnetic stirring bar. The concentrations ofO-palmitoyl-heparin DS2 and of α-cyclodextrin are shown in Table 7. Theresults of the measurements of the hydrodynamic diameters are shown inTable 7 and FIG. 17.

TABLE 7 Effect of varying the concentration of α-CD on the hydrodynamicdiameter of the nanoparticles consisting of heparin DS2 Concen- Concen-Concen- tration of tration tration O-palmitoyl- ratio α-CD/O- of α-CDheparin palmitoyl- Name (g/L) DS2 (g/L) heparin DS2 D_(h) (nm) Hep5 0 100 No formation of particles Hep6 100 10 10 340 ± 19 Hep7 50 10 5 355 ±11 Hep8 25 10 2.5 410 ± 24

FIG. 18 shows an example of images obtained by observations usingtransmission electron microscopy of the nanoparticles consisting ofheparin DS2. These images, obtained without contrast agents, show thatthese nanoparticles self-assemble in a well-structured manner in theform of a hexagon.

Example 17 Preparation and Characterization of Microparticles ofO-Palmitoyl-Carrageenan DS1

O-Palmitoyl-carrageenan DS1 as well as α-cyclodextrin are weighed in aflask. Next, water is added to the mixture. The whole is mixed for 72 hat ambient temperature using a magnetic stirring bar The concentrationsof O-palmitoyl-carrageenan and of α-cyclodextrin, and the results of themeasurements of the hydrodynamic diameters, are shown in Table 8.

TABLE 8 Effect of varying the concentration of α-CD on the hydrodynamicdiameter of the microparticles consisting of carrageenan DS1 Variationin the quantity of α-cyclodextrin (FIG. 19) Concen- Concen- Concen-tration of tration tration O-palmitoyl- ratio α-CD/O- of α-CDcarrageenan palmitoyl- Name (g/L) DS1 (g/L) carrageenan DS1 D_(h) (nm)Carr1 0 10 0 No formation of particles, the product is insolu- ble inwater Carr2 100 10 10 1196 ± 242 Carr3 51 10 5 2053 ± 642 Carr4 27 102.7 3613 ± 800

TABLE 9 Effect of varying the concentration of O-palmitoyl-carrageenanon the hy- drodynamic diameter of the microparticles consisting ofcarrageenan DS1 Variation in the quantity of O-palmitoyl-carrageenan DS1(FIG. 20) Concen- Concen- Concen- tration of tration trationO-palmitoyl- ratio α-CD/O- of α-CD carrageenan palmitoyl- Name (g/L) DS1(g/L) carrageenan DS1 D_(h) (nm) Carr5 100 2.5 40 1089 ± 272 Carr6 100 520 1277 ± 218 Carr7 100 10 10 1196 ± 242 Carr8 100 20 5 1384 ± 225 Carr9100 30 3.3 1658 ± 403

Example 18 Preparation and Characterization of Microparticles andNanoparticles of O-Palmitoyl-Carrageenan DS2

O-Palmitoyl-carrageenan DS2 and α-cyclodextrin are weighed in a flask.Next, water is added to the mixture. The whole is mixed for 72 h atambient temperature using a magnetic stirring bar. The concentrations ofO-palmitoyl-carrageenan and of α-cyclodextrin, and the results of themeasurements of the hydrodynamic diameters, are shown in Table 10.

TABLE 10 Effect of varying the concentration of α-CD on the hydrodynamicdiame- ter of the microparticles and nanoparticles composed ofcarrageenan DS2 Variation in the quantity of α-cyclodextrin (FIG. 21)Concen- Concen- Concen- tration of tration tration O-palmitoyl- ratioα-CD/O- of α-CD carrageenan palmitoyl- Name (g/L) DS2 (g/L) carrageenanDS2 D_(h) (nm) Carr10 0 10 0 No formation of particles, the product isinsolu- ble in water Carr11 100 10 10 3409 ± 226 Carr12 50 10 5 2804 ±288 Carr13 25 10 2.5 1412 ± 290 Carr14 10 10 1 1129 ± 491 Carr15 5 100.5  365 ± 114

Example 19 Preparation and Characterization of Microparticles ofO-Palmitoyl-Carrageenan DS3

O-Palmitoyl-carrageenan DS3 and α-cyclodextrin are weighed in a flask.Next, water is added to the mixture. The whole is mixed for 72 h atambient temperature. The concentrations of O-palmitoyl-carrageenan andof α-cyclodextrin, and the results of the measurements of thehydrodynamic diameters, are shown in Table 11.

TABLE 11 Effect of varying the concentration of O-palmitoyl-carrageenanon the hy- drodynamic diameter of the microparticles composed ofcarrageenan DS3 Variation in the quantity of O-palmitoyl-carrageenanDS3. Concen- Concen- Concen- tration of tration ratio trationO-palmitoyl- α-CD/O- of α-CD carrageenan palmitoyl- Name (g/L) DS3 (g/L)carrageenan DS3 D_(h) (nm) Carr16 100 2.5 40 1457 ± 431 Carr17 100 5 202521 ± 612 Carr18 100 10 10 3340 ± 265 Carr19 100 20 5 5011 ± 371 Carr20100 30 3.3 6125 ± 730

Example 20 Manufacture of Microparticles and Nanoparticles Composed ofN-Palmitoyl-Chitosan in the Presence of Castor Oil

The microparticles and nanoparticles were formed from α-cyclodextrin andN-palmitoyl-chitosan MC9. The concentrations used are presented in thetable below. The protocol consists of weighing N-palmitoyl-chitosan,α-cyclodextrin and castor oil labelled beforehand with Sudan III. Theaddition of Sudan III makes it possible to detect the phenomena ofinstability of the preparations. Next, water is added to the mixture.The whole is mixed for 72 h at ambient temperature.

The preparations observed by eye did not show phenomena of instability.The results of the measurements of size are presented in the followingtable.

TABLE 12 Effect of varying the concentration of castor oil on thehydrodynamic diameter of the microparticles and nanoparticles composedof chitosan MC9 Concentration Concentration of Concentration of of α-CD(g/L) MC9 DS 17.01% (g/L) castor oil (g/L) D_(h) (nm) 100 25 0 1416 ±45  100 25 13 896 ± 44 100 25 23 964 ± 31

Example 21 Manufacture of Microparticles Composed of O-Oleoyl-Chitin inthe Presence of Castor Oil

The microparticles were formed from α-cyclodextrin and O-oleoyl-chitinDS 0.68%. The concentrations used are shown in the table below. Theprotocol consists of weighing O-oleoyl-chitin, α-cyclodextrin and castoroil labelled beforehand with Sudan III. The addition of Sudan III makesit possible to detect the phenomena of instability of the preparations.Next, water is added to the mixture. The whole is mixed for 72 h atambient temperature. The preparations observed by eye did not showphenomena of instability. The results of the measurements of size arepresented in the following table.

TABLE 13 Effect of varying the concentration of castor oil and theconcentration of O-oleoyl-chitin on the hydrodynamic diameter of themicroparticles Concentration of Concentration O-oleoyl-chitinConcentration of of α-CD (g/L) DS 0.68% (g/L) castor oil (g/L) D_(h)(nm) 100 25 0  2734 ± 206 100 25 13 2057 ± 54 100 25 19.5  1875 ± 121100 50 0 1487 ± 58 100 50 12 1579 ± 98 100 50 19.5 1310 ± 38

Example 22 Absence of the Formation of Particles Using Native Chitosan,not Grafted with Palmitic Acid

α-Cyclodextrin (100 mg) and native chitosan (10 mg) were weighed in avial. Next, water is added to the mixture to give a total volume of 1mL. The whole is mixed using a magnetic stirring bar for 72 h at ambienttemperature. FIG. 23 (1) is an image obtained after observations usingtransmission electron microscopy and shows the absence of formation ofnanoparticles or of microparticles in comparison with the images of thenanoparticles obtained with O-palmitoyl-chitosanMC6/α-cyclodextrin/water (1/10/89)%.

Example 23 Drying of the Particles by Lyophilization

Lyophilization is a method of drying under vacuum at low temperature.The product containing water is frozen beforehand. Lyophilization isstill the method of choice for drying heat-sensitive products.

The microparticles were prepared from α-cyclodextrin andN-oleoyl-chitosan, MC4, DS 13.47%. The protocol consists of weighingN-oleoyl-chitosan and α-cyclodextrin. The concentrations used are givenin Table 14. Next, water is added to the mixture. The whole is mixed for72 h at ambient temperature.

Lyophilization is carried out on 1 mL of preparation containing themicroparticles, frozen beforehand at (−20° C.) for 12 h, and then placedin the lyophilizer for 24 h.

After being lyophilized, the preparations are resuspended in 1 mL ofMilliQ® water; it is noted that the macroscopic appearance of thesamples has not changed. In order to ensure that the lyophilization stephad not altered the physicochemical characteristics of the particles,measurements of the hydrodynamic diameter were carried out afterlyophilization and were compared with those obtained beforelyophilization (Table 14).

TABLE 14 Measurement of the hydrodynamic diameters of the particles (ofchitosan MC4: 145 kDa N-acylated with OA and a DS 13.47%) before andafter lyophilization Concentration D_(h) (nm) of N-oleoyl- ConcentrationBefore After chitosan (g/L) of α-CD (g/L) lyophilization lyophilization10 20 3050 ± 345 2880 ± 1102 10 100 1300 ± 139 1910 ± 173 

Example 24 Scale-Up of Particle Manufacture

Transition to the pilot scale is a necessity for developing this methodon an industrial scale. A pilot plant was designed composed of a reactorstirred with a mechanical stirrer of the propeller type. The stirringspeed is fixed at 350 rpm. Regulation of the temperature at 25° C. isensured by circulation of fluid (water/ethylene glycol) connected to athermostat. 100-mL batches of microparticles are obtained by addingsuccessively to the reactor (1 g) of chitosan of molecular weight 250kDa N-acylated with 10 equivalents of palmitic acid (MC9, DS 17.01%), 10g of α-cyclodextrin and MilliQ® water q.s. 100 mL.

The results obtained at the pilot scale are presented in Table 15 andcompared with those obtained at the laboratory scale.

TABLE 15 Results of measurements of the hydrodynamic diameters of themicroparticles obtained at the pilot scale in comparison with thoseobtained at the laboratory scale Laboratory scale Pilot scale Finalvolume 2 mL 100 mL prepared D_(h) (nm) 1680 ± 437 1730 ± 110

No significant change in D_(h) was reported. The TEM images showed thatthe microparticles retained their hexagonal structure.

Example 25 Manufacture of Microparticles with a Mixture of TwoPolysaccharides

TABLE 16 Hydrodynamic diameters of the microparticles composed of amixture of O-palmitoyl-chitosan and O-palmitoyl-dextran ConcentrationConcentration Concentration of O-palmitoyl- of O-palmitoyl- of α-CD(g/L) chitosan (g/L) dextran (g/L) D_(h) (nm) 100 7.6 2.5 1707 ± 122 1002.5 7.5 3261 ± 230

TABLE 17 Hydrodynamic diameters of the microparticles composed of amixture of O-palmitoyl-chitosan and O-palmitoyl-pullulan ConcentrationConcentration Concentration of O-palmitoyl- of O-palmitoyl- of α-CD(g/L) chitosan (g/L) pullulan (g/L) D_(h) (nm) 100 7.7 2.5 2292 ± 89 100 5 5 2908 ± 112 100 2.5 7.5 2057 ± 271

TABLE 18 Hydrodynamic diameters of the microparticles composed of amixture of O-palmitoyl-chitin and O-palmitoyl-dextran ConcentrationConcentration Concentration of O-oleoyl- of O-palmitoyl- of α-CD (g/L)chitin (g/L) dextran (g/L) D_(h) (nm) 100 7.6 2.5 4899 ± 185 100 2.5 7.53960 ± 311

TABLE 20 Hydrodynamic diameters of the microparticles composed of amixture of O-palmitoyl-chitin and O-palmitoyl-pullulan ConcentrationConcentration Concentration of O-oleoyl- of O-palmitoyl- of α-CD (g/L)chitin (g/L) pullulan (g/L) D_(h) (nm) 100 5 5 3372 ± 283 100 2.5 7.53342 ± 210

Example 26 Antiviral Activity of the Nanoparticles Composed ofO-Palmitoyl Heparin

Cells. Kidney epithelial cells extracted from an African green monkey(Vero cells, (ATCC CCL-81), human epithelial cells Hep-2 and (ATCCCCL-23) and kidney epithelial cells extracted from an African greenmonkey (MA-104, ATCC CRL-2378.1) are cultured in monolayers in Eagle'sminimum essential medium (MEM) (Gibco/BRL, Gaithersburg, Md.)supplemented with 10% of heat-inactivated fetal calf serum and 1% ofantibiotic-antifungal solution (Zell Shield, Minerva Biolabs GmbH,Berlin, Germany). The 293TT cell line, derived from human embryonickidney cells transformed with the large T antigen of simian virus 40(SV40), is cultured in Dulbecco modified Eagle medium (DMEM) (Gibco-BRL,Gaithersburg, Md.) supplemented with 10% of fetal calf serum (FCS;Gibco-BRL), Glutamax-I 1% (Invitrogen, Carlsbad, Calif.) and 1% ofnon-essential amino acids (Sigma Aldrich, Steinheim, Germany). The 293TTcells allow expression of a high level of proteins from vectorscontaining the replication origin of SV40 owing to over-replication ofthe expression vectors (Buck et al., 2004).

Virus. Clinical isolates of HSV-1 and HSV-2 were supplied by ProfessorM. Pistello (University of Pisa, Italy). The HSV-1 and HSV-2 strainswere propagated and titrated by the plating technique on Vero cells. Thestrain A2 RSV (ATCC VR-1540) was propagated and titrated by theReed-Muench method on Hep-2 cells described previously (Donalisio etal., 2012). The strain of human rotavirus Wa(ATCC VR-2018) was activatedwith 5 μg/ml of porcine pancreatic trypsin of the IX type (Sigma, St.Louis, Mo.) for 30 min at 37° C. and propagated in MA104 cells using MEMmedium containing 0.5 μg of trypsin per ml as described previously(Coulson et al., 1986). The stocks of virus were kept frozen (−80° C.).

HPV PsV production. The plasmids and 293TT cells used for producingpseudovirus (PsV) were supplied by John Schiller (National CancerInstitute, Bethesda, Md.). The details of the protocols and the maps ofthe plasmids of this study may be consulted on the following link:http://home.ccr.cancer.gov/lco/default. asp. HPV-16 pseudoviruses wereproduced by methods described previously (Buck et al., 2005). Briefly,the 293TT cells are transfected with plasmids expressing the proteins ofmajor and minor capsids of papillomaviruses (respectively L1 and L2) anda reporter plasmid expressing secreted alkaline phosphatase (SEAP),pYSEAP. The capsids were matured overnight in a cellular lysate, and theclarified supernatant was then loaded above a density gradient from 27to 33 to 39%, Optiprep (Sigma-Aldrich, St. Louis, Mo.), at ambienttemperature for 4 h. The material was then centrifuged at 340,000 g for3.5 h at 16° C. in an SW50.1 rotor (Beckman Coulter, Inc., Fullerton,Calif.) and then collected by puncture at the bottom of the tubes. Thefractions are analysed for purity in gels of glycine-Tris-10% SodiumDodecyl Sulphate (SDS), titrated on 293TT cells to test infectivity bydetection with SEAP, and then combined and frozen at −80° C. for thedesired time. The content of protein L1 of the stocks of PsV wasdetermined by comparison with standard bovine serum albumin onpolyacrylamide-SDS gels stained with Coomassie Blue.

TABLE 21 Values of the median effective concentration (EC50) in μg/mL.Hep2 Hep3 Hep4 Hep8 HSV-1 0.99 1.25 1.13 6.61 HSV-2 0.65 0.83 1.22 3.65HPV-16 1.96 0.24 3.95 2.55 RSV 0.24 0.28 0.27 0.36 Rotavirus — — — —

Example 27 Absence of the Formation of Particles Using the Derivativesof β-Cyclodextrin and O-Palmitoyl-Chitosan (MC12)

TABLE 22 Absence of the formation of particles by mixing O-palmitoyl-chitosan (MC12) with HP-β-CD or Me-β-CD Concentration ConcentrationAppearance of the CD used of MC12 (g/L) of CD (g/L) preparation HP-β-CD1.25 100 Large insoluble white 2.5 100 aggregates 5 100 5 10 5 15 5 20 525 5 50 Me-β-CD 2.5 100 5 100 10 100 5 10 5 15 5 20 5 25 5 50

Example 28 Absence of the Formation of Particles Using the Derivativesof β-Cyclodextrin and O-Oleoyl-Chitin

TABLE 23 Absence of the formation of particles using the derivatives ofβ-cyclodextrin and O-oleoyl-chitin Concentration O-oleoyl-chitinConcentration Appearance of the CD used DS 0.1% (g/L) CD (g/L)preparation HP-β-CD 2.5 100 Large white aggregates 5 100 10 100 5 10 515 5 20 5 25 Me-β-CD 2.5 100 White aggregates 5 100 10 100 5 10 5 15 520 5 25 5 50

Example 29 Absence of the Formation of Particles Using the Derivativesof β-Cyclodextrin and O-Palmitoyl-Pullulan

TABLE 24 Absence of the formation of particles using the derivatives ofβ-cyclodextrin and O-palmitoyl-pullulan Concentration of O-palmitoyl-Concentration Appearance of the CD used pullulan (g/L) CD (g/L)preparation HP-β- 2.5 100 Solution: dissolution of CD 5 100O-palmitoyl-pullulan 10 100 White aggregates 5 10 5 15 5 20 5 25 Me-β-2.5 100 CD 5 100 10 100 5 10

Example 30 Preparation and Characterization of Microparticles ofO-Palmitoyl-Hyaluronic Acid

O-Palmitoyl-hyaluronic acid and α-cyclodextrin are weighed in a flask.Next, water is added to the mixture. The whole is mixed for 72 h atambient temperature. The concentration of α-cyclodextrin and the resultsof the measurements of the hydrodynamic diameters are shown in Table 25.

TABLE 25 Effect of varying the concentration of α-CD on the size of themicroparticles consisting of O-palmitoyl-hyaluronic acid ConcentrationConcentration of O-palmitoyl- ratio α-CD/ Concentration hyaluronicO-palmitoyl- of α-CD (g/L) acid (g/L) acid hyaluronic D_(h) (nm) 0 10 0Absence of the formation of particles, presence of large insolubleaggregates 25 10 2.5 1946 ± 589 100 10 10 2479 ± 334

Example 31 Encapsulation of the Active Ingredients in the Presence of aSolvent

The protocol adopted for encapsulating the hydrophobic activeingredients is to dissolve the active ingredient in an ethanol/watermixture at an initial concentration as shown in Table 26 and then addthe amphiphilic polysaccharide and the α-cyclodextrin. The mixture ismaintained under magnetic stirring for 3 days. After mixing for 3 days,the concentration of the active ingredient that has not beenencapsulated is determined in the supernatant of the preparation. Thesupernatant is separated either by simple sedimentation or bycentrifugation of the microparticles, or after ultracentrifugation inthe case of the nanoparticles.

TABLE 26 Example of encapsulated active molecules. Initial Encapsu- Polyconcentration lated Encapsu- Ethanol/ saccha- of active concen- lationwater ride molecules tration yield % v/v used (g/L) (g/L) (%) Ketoprofen40/60 MC6 3.5 1.09 31.12

1. Inclusion complex formed by interaction between at least onepolysaccharide selected from chitosan, dextran, hyaluronic acid,amylose, amylopectin, pullulan, heparin, chitin, heparan sulphate,dermatan sulphate, keratan sulphate, chondroitin sulphate, cellulosesulphate, dextran sulphate, dextrin sulphate, starch, pectin, thealginates, the carrageenans, fucan, curdlan, xylan, polyguluronic acid,xanthan, arabinan, polymannuronic acid, and derivatives thereof, saidpolysaccharide comprising hydrophobic groups selected from linear orbranched alkyl groups containing from 2 to 1000 carbon atoms, or linearor branched, in particular linear, alkenyl groups, which may contain atleast one C═C double bond, said hydrophobic groups being boundcovalently to said polysaccharide, and one α-cyclodextrin (CD) in theform of monomer, the polysaccharide and the cyclodextrin being boundnon-covalently.
 2. Inclusion complex according to claim 1, wherein thepolysaccharide is composed of at least 3 saccharide units, its molecularweight being in particular in the range from 100 Da to 1,000,000 kDa,and in particular is equal to 20 kDa, 145 kDa or 250 kDa, and inparticular wherein the degree of substitution of the polysaccharide withthe hydrophobic groups is from 0.001 to 100%, in particular from 0.05 to50%.
 3. Inclusion complex according to claim 1, wherein the ratio of theconcentration of cyclodextrin in g/L to that of the polysaccharide is inthe range from 10⁻⁶ to 900,000, in particular in the range from 4 to 15,and in particular is equal to
 10. 4. Inclusion complex according toclaim 1, wherein: the polysaccharide is a chitosan bearing hydrophobicgroups at the level of oxygen atoms originating from the —OH groupsand/or from the —CH₂OH groups fixed to the chitosan ring, and having theformula

in which R represents a hydrogen atom, or a group of formula

in which R⁴ represents the hydrophobic group and is selected from: alinear or branched alkyl group containing from 1 to 1000 carbon atoms,in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃ groups, a linear orbranched alkenyl group containing 2 to 1000 carbon atoms and containingat least one C═C double bond, in particular the—(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₃ or —(CH₂)₇—CH═CH—(CH₂)₇—CH₃ groups,provided that R represents at least one group

of formula m represents the number of D-glucosamine units, n representsthe number of N-acetyl-D-glucosamine units, provided that the degree ofdeacetylation (DDA) representing the percentage of m relative to thetotal number of units is greater than 50%, and the α-cyclodextrin CD hasthe formula

in which p is equal to 6, R¹, R² and R³, which may be identical ordifferent, in particular identical, are hydrogen atoms, alkyl groupscomprising 1 to 3 carbon atoms, selected from methyls, ethyls, propyls,isopropyls, —NH₂ amino groups, —NH₃ ⁺ ammonium groups, or —SO₄ ²⁻sulphate groups, and are in particular hydrogen atoms or methyl groups,said CD being alpha-cyclodextrin (α-CD) in the form of monomer, and inparticular wherein the cyclodextrin is functionalized with a ligandselected from antibodies, antibody fragments, receptors, lectins, biotinor derivatives thereof.
 5. Inclusion complex according to claim 1,wherein the polysaccharide is a dextran bearing hydrophobic groups,fixed by oxygen atoms of said dextran and representing groups of formula

in which R⁴ has the meanings designated above, * represents dextran, thecyclodextrin is α-CD, or wherein the polysaccharide is hyaluronic acidbearing hydrophobic groups, fixed by oxygen atoms of said hyaluronicacid and representing groups of formula

in which R⁴ has the meanings designated above, * represents hyaluronicacid, the cyclodextrin is α-CD, or wherein the polysaccharide is anamylopectin bearing hydrophobic groups fixed by oxygen atoms of saidamylopectin and representing groups of formula

in which R⁴ has the meanings designated above, * represents anamylopectin, the cyclodextrin is α-CD, or wherein the polysaccharide isa pullulan bearing hydrophobic groups fixed by oxygen atoms of saidpullulan and representing groups of formula

in which R⁴ has the meanings designated above, * represents pullulan,the cyclodextrin is cx-CD, or wherein the polysaccharide is a heparinbearing hydrophobic groups fixed by nitrogen atoms of said heparin andrepresenting groups of formula

in which R⁴ has the meanings designated above, * represents heparin, orwherein the polysaccharide is a heparin bearing hydrophobic groups fixedby oxygen atoms of said heparin, these oxygens possibly originating fromthe hydroxyl or carboxyl groups of the heparin, and representing groupsof formula

in which R⁴ has the meanings designated above, * represents heparin, andthe cyclodextrin is α-CD, or wherein the polysaccharide is a carrageenanbearing hydrophobic groups fixed by nitrogen atoms of said carrageenansulphate and representing groups of formula

in which R⁴ has the meanings designated above, * represents carrageenan,or the polysaccharide is a carrageenan bearing hydrophobic groups fixedby oxygen atoms of said carrageenan, these oxygens possibly originatingfrom the hydroxyl or carboxyl groups of carrageenan, and representinggroups of formula

in which R⁴ has the meanings designated above, * represents carrageenan,and the cyclodextrin is α-CD.
 6. Particle with a size in the range from1 nm to 100,000 nm containing inclusion complexes according to claim 1by interaction between at least one polysaccharide selected fromchitosan, dextran, hyaluronic acid, amylose, amylopectin, pullulan,heparin, chitin, cellulose derivatives, heparan sulphate, dermatansulphate, keratan sulphate, chondroitin sulphate, cellulose sulphate,dextran sulphate, dextrin sulphate, starch, pectin, the alginates, thecarrageenans, fucan, curdlan, xylan, polyguluronic acid, xanthan,arabinan, polymannuronic acid, and derivatives thereof, comprisinghydrophobic groups selected from linear or branched alkyl groups orlinear or branched alkenyl groups bearing from 1 to 4 C═C double bonds,conjugated or not, said hydrophobic groups being bound covalently tosaid polysaccharide, optionally functionalized with a ligand selectedfrom antibodies, antibody fragments, receptors, lectins or biotin orderivatives thereof, an α-cyclodextrin in the form of monomer,optionally functionalized with a ligand selected from antibodies,antibody fragments, receptors, lectins or biotin or derivatives thereof.7. Encapsulation system containing one or more particles according toclaim 6, and a substance used for its properties in the pharmaceutical,medical, paramedical, medical-device, cosmetic, veterinary, agri-food,animal-feed, agrochemical, pesticide, cosmetotextile, perfumery, andenvironmental fields, in particular water purification, in the paint,building and/or car industry.
 8. Pharmaceutical composition containing,as active substance, a substance encapsulated in inclusion complexesaccording to claim 1 or in particles, with a size in the range from 1 nmto 100,000 nm containing said inclusion complexes by interaction betweenat least one polysaccharide selected from chitosan, dextran, hyaluronicacid, amylose, amylopectin, pullulan, heparin, chitin, cellulosederivatives, heparan sulphate, dermatan sulphate, keratan sulphate,chondroitin sulphate, cellulose sulphate, dextran sulphate, dextrinsulphate, starch, pectin, the alginates, the carrageenans, fucan,curdlan, xylan, polyguluronic acid, xanthan, arabinan, polymannuronicacid, and derivatives thereof, comprising hydrophobic groups selectedfrom linear or branched alkyl groups or linear or branched alkenylgroups bearing from 1 to 4 C═C double bonds, conjugated or not, saidhydrophobic groups being bound covalently to said polysaccharide,optionally functionalized with a ligand selected from antibodies,antibody fragments, receptors, lectins or biotin or derivatives thereof,an α-cyclodextrin in the form of monomer, optionally functionalized witha ligand selected from antibodies, antibody fragments, receptors,lectins or biotin or derivatives thereof, together with apharmaceutically acceptable vehicle, in solid form, or in the form ofsolution or suspension in a physiological medium, usable by thefollowing routes: parenteral, oral, cutaneous, subcutaneous, nasal,pulmonary or ocular, and for any administration at the level of a mucousmembrane, in particular in the form of pills (tablets), soft capsules,hard capsules (gelatin capsules), powders, granules, soluble ordispersible tablets, patches, implants, suppositories, solutions,suspension, syrup, pastes, creams, gels, emulsions, sprays, lotions, orointments.
 9. Method for preparing an inclusion complex according toclaim 1, comprising a step of mixing at least: one polysaccharide in theform of suspension in a solvent, in particular water comprisinghydrophobic groups bound covalently to the polysaccharide by oxygenatoms of said polysaccharide, and one α-cyclodextrin (CD) in the form ofmonomer, preferably in suspension in a solvent, in particular water, toobtain an inclusion complex, wherein said polysaccharide and saidcyclodextrin are bound non-covalently, and in particular comprising astep of mixing a polysaccharide selected from chitosan, dextran,hyaluronic acid, amylose, amylopectin, pullulan, heparin, chitin,heparan sulphate, dermatan sulphate, keratan sulphate, chondroitinsulphate, cellulose sulphate, dextran sulphate, dextrin sulphate,starch, pectin, the alginates, the carrageenans, fucan, curdlan, xylan,polyglucuronic acid, xanthan, arabinan, polymannuronic acid, andderivatives thereof, said polysaccharide comprising hydrophobic groupsof formula

in which * represents the polysaccharide, R⁴ represents a linear orbranched alkyl group containing from 1 to 1000 carbon atoms, inparticular the —(CH₂)₂₄—CH₂ or —(CH₂)₁₆—CH₂ groups, a linear or branchedalkenyl group containing 2 to 1000 carbon atoms and bearing at least oneC═C double bond, in particular the —(CH₂)₇—CH═CH—CH₂—(CH₂)₇—CH₂ or—(CH₂)₇—CH—CH—(CH₂)₇—CH₃ groups, an α-cyclodextrin (CD) having theformula

in which p is equal to 6, R¹, R² and R³, which may be identical ordifferent, in particular identical, are hydrogen atoms, alkyl groupscomprising 1 to 3 carbon atoms, selected from methyls, ethyls, propyls,isopropyls, —NH₂ amino groups, —NH₃ ⁺ ammonium groups, or —SO₄ ²⁻sulphate groups, and are in particular hydrogen atoms or methyl groups,said CD being alpha-cyclodextrin (α-CD) in the form of monomer, toobtain an inclusion complex, wherein said polysaccharide and saidcyclodextrin are bound non-covalently.
 10. Polysaccharide selected from:a chitosan bearing hydrophobic groups having formula II

a carrageenan bearing hydrophobic groups having formula I and/or II

a pullulan bearing hydrophobic groups having the formula

a hyaluronic acid bearing hydrophobic groups having the formula

a chitin bearing hydrophobic groups having the formula

in which R represents a hydrogen atom, or a group of formula

in which R⁴ represents a linear or branched alkyl group containing from1 to 1000 carbon atoms, in particular the —(CH₂)₁₄—CH₃ or —(CH₂)₁₆—CH₃groups, a linear or branched alkenyl group containing 2 to 1000 carbonatoms and bearing at least one C═C double bond, in particularthe—(CH₂)₇—CH═CH—CH₂— (CH₂)₇—CH₃ or—(CH₂)₇—CH═CH— (CH₂)₇—CH₃ groups,provided that R represents at least one group of formula

m represents the number of N-glucosamine units, n represents the numberof N-acetyl-glucosamine units, provided that the percentage of units nrelative to the total number of units is greater than 50%.
 11. A methodfor vaccination, treatment of burns, and/or for wound-healing comprisingadministering to a subject in need thereof a therapeutically effectiveamount of particles according to claim
 6. 12. A method for preventing,inhibiting and/or treating fungal, bacterial, viral and/or parasiticinfections on biotic or abiotic surfaces, applying to said surfaces aneffective amount of the particles according to claim
 6. 13. Veterinarymedicaments, in particular as adjuvants for vaccination, comprisingparticles according to claim
 6. 14. A cosmetic agent, in particular asanti-ageing, depigmenting, wound-healing, hydrating, perfuming,deodorizing, antiperspirant, cleaning, colouring, or preserving agent,comprising particles according to claim
 6. 15. A method for preparingdevices, in particular wound-healing dressings, said devices comprisingparticles according to claim 6, and being able to release said particlesor one or more active substance(s) of interest contained in saidparticles.